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A pyrene-based fluorescent sensor for ratiometric detection of heparin and its complex with heparin for reversed ratiometric detection of protamine in aqueous solution
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 170 (2017) 198–205
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A pyrene-based fluorescent sensor for ratiometric detection of heparin and its complex with heparin for reversed ratiometric detection of protamine in aqueous solution Weiwei Gong, Shihuai Wang, Yuting Wei, Liping Ding ⁎, Yu Fang Key Laboratory of Applied Surface and Colloid Chemistry of Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, PR China
a r t i c l e
i n f o
Article history: Received 20 April 2016 Received in revised form 13 July 2016 Accepted 14 July 2016 Available online 16 July 2016 Keywords: Heparin Protamine Excimer Fluorescence Serum solution
a b s t r a c t An imidazolium-modified pyrene derivative, IPy, was used for ratiometric detection of heparin, and its complex with heparin was used for reversed ratiometric detection of protamine in both aqueous solution and serum samples. The cationic fluorescent probe could interact with anionic heparin via electrostatic interaction to bring about blue-to-green fluorescence changes as monomer emission significantly decreases and excimer increases. The binary combination of IPy and heparin could be further used for green-to-blue detection of protamine since heparin prefers to bind to protamine instead of the probe due to its stronger affinity with protamine. The cationic probe shows high sensitivity to heparin with a low detection limit of 8.5 nM (153 ng/mL) and its combination with heparin displays high sensitivity to protamine with a detection limit as low as 15.4 nM (107.8 ng/mL) according to the 3σ IUPAC criteria. Moreover, both sensing processes are fast and can be performed in serum solutions, indicating possibility for practical applications. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Heparin is a linear polysaccharide with abundant of sulfate and carboxylate groups, and therefore has the highest negative charge density among any known biological macromolecules [1,2]. Heparin plays an important role in regulating various biological processes, such as cell growth and differentiation, inflammation, and blood coagulation [3,4]. More importantly, heparin has been widely used in clinical applications such as preventing thrombosis during surgery and as an anticoagulant drug to treat thrombotic diseases [5]. However, overdose of heparin can induce some complications such as hemorrhages and heparin-induced thrombocytopenia [3]. The suggested therapeutic dose of heparin is 2–8 U/mL (17–67 μM) during cardiovascular surgery and 0.2–1.2 U/mL (1.7–10 μM) for postoperative and long-term care, respectively [6,7]. Protamine, as a well-known heparin antidote, is a highly cationic protein (pI = 13.8) with high content of basic arginine residues. The anticoagulant effect of heparin can be reversed by protamine due to their combination through electrostatic interaction [8]. Thus, protamine is often used for treatment of heparin overdose. However, the over-use of protamine could also induce adverse effect such as hypotension and idiosyncratic fatal cardiac arrest [9]. Therefore, the detection and quantification of both heparin and protamine are of crucial significance for clinical procedures.
⁎ Corresponding author. E-mail address: [email protected] (L. Ding).
http://dx.doi.org/10.1016/j.saa.2016.07.026 1386-1425/© 2016 Elsevier B.V. All rights reserved.
Among various methods including colorimetric sensor [10,11], electrochemical sensor [4], and surface-enhanced Raman sensor [12], fluorescent sensors have drawn extensive attention because of their advantages in terms of high sensitivity, high selectivity, easy operation and real time detection [13]. Up to now, a great amount of fluorescent sensors have been developed for detecting heparin and protamine. These sensors exhibit either turn-off responses [2,14–16] or turn-on [17–19] or ratiometric responses [3,5,20,21] to the presence of the target analyte. The turn-off and turn-on sensors rely on fluorescence intensity variation at one wavelength and are easily affected by instrumental and environmental conditions. By comparison, ratiometric fluorescent sensors are advantageous because they usually depend on the intensity ratio at two emission wavelengths, which can help avoiding the abovementioned interferences [22]. Most of the above mentioned fluorescent sensors detect only one target, either heparin or protamine. Very recently, fluorescent sensors for recognizing both heparin and protamine are attracting attention since they are more convenient and multi-functional. Zhang and coworkers used gold nanoparticles coupled with fluorescent emitting materials such as silicon quantum dots [23], fluorescein [24], and upconversion nanoparticles [25] for detecting both protamine and heparin. These gold nanoparticle-attached fluorescent materials are fluorescence inert due to the quenching effect of gold surface, and show turn-on responses to protamine as protamine interacts with gold nanoparticles and de-attaches fluorescent materials from gold surface, and then show turn-off responses to heparin because heparin has stronger affinity with protamine that results in binding fluorescent materials
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with gold nanoparticles again. John et al. reported using folic acid capped gold nanoparticles for detecting both protamine and heparin based on aggregation and de-aggregation of gold nanoparticles induced by the added analyte [26]. Su et al. developed a fluorescein-labeled DNA probe for turn-off sensing protamine and turn-on sensing heparin [8]. Although these sensors show some interesting sensing behaviors, they have some drawbacks like long time of incubation [23–25], or low sensitivity [9]. Moreover, they are all based on single signal of fluorescence variation at one wavelength. Fluorescent ratiometric sensors for detecting both heparin and protamine have never been reported, to our knowledge. In the present study, we reported using a cationic pyrene-based fluorophore for ratiometric detection of both heparin and protamine in total aqueous solution. Pyrene-based fluorophores have been widely used to develop ratiometric sensors as they can emit monomer and excimer emission [27–29], and more recently used for ratiometric detection of various biomolecules such as G-quadruplex structures of Grich DNA [30], human breast cancer cell [31], and proteins [32]. Their fluorescence emission is highly dependent on the conformation or aggregation of pyrene derivatives and very sensitive to the microenvironment. Although a variety of pyrene derivatives were reported for ratiometric sensing heparin [3,5,21,33,34], they were not used to detect protamine or both targets. The present imidazolium-modified pyrene derivative (IPy, Scheme 1) exhibits only monomer emission in aqueous buffer solution, and shows blue-to-green emission change upon addition of heparin as monomer emission significantly decreases accompanied by excimer emission increasing. The binary combination of IPy and heparin displays green-to-blue changes to the addition of protamine as witnessed by recovered monomer emission and decreased excimer emission. Moreover, both sensing processes show high sensitivity and can be performed in serum solutions.
2. Experimental methods 2.1. Materials and instruments The pyrene derivative (IPy) was synthesized and characterized according to our previously reported method [35]. Heparin sodium salt (from hog intestine) was purchased from TCI. N-(2-hydroxyethyl) piperazine-Nʹ-ethanesulfonic acid (HEPES), chondroitin sulfate sodium salt, adenosine 5ʹ-triphosphate disodium salt hydrate (ATP, ≥99%), cytidine 5ʹ-triphosphate disodium salt (CTP, ≥95%), uridine 5ʹ-triphosphate trisodium salt hydrate (UTP, ≥96%), L-arginine (99%), L-lysine (99%), Lhistidine (99%) and all proteins including protamine (from salmon), bovine serum albumin (≥ 98%), lysozyme (from chicken egg, ≥ 90%), trypsin (from bovine pancreas), pepsin (from porcine gastric mucosa), β-lactoglobulin (from bovine milk, ≥90%), cytochrome c (from bovine heart, ≥95%), ovalbumin egg (from chicken egg white, ≥ll p, hyaluronic acid sodium salt, and poly-L-lysine hydrobromide were purchased from Sigma-Aldrich. HIV-1 TAT peptide (Sequence: YGRKKRRQRRR) was obtained from Chinese Peptide Company. Fetal bovine serum was
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purchased from Zhejiang Tianhang Biotechnology Co. Ltd. All chemicals were used as received. Steady-state fluorescence spectra were measured on a single photon-counting fluorescence spectrometer (FS5, Edinburgh Instruments) with xenon light (150 W) as the excitation source, and the excitation and emission slit widths were set at 2.1 and 0.3 nm, respectively. All samples were excited at 346 nm. Time-resolved fluorescence decays were measured on a time-resolved single photon-counting fluorescence spectrometer (FS920, Edinburgh Instruments) with 343.4 nm laser as the excitation source. UV–vis absorption spectra were recorded on a spectrophotometer (U3900, Hitachi Instrument). 2.2. Preparation of samples The aqueous stock solution of HEPES (100 mM) was first prepared by dissolving solid HEPES in neat water and using NaOH (1.0 M) to regulate pH to 7.4. HEPES buffer solution (10 mM, pH 7.4) was prepared by diluting the stock HEPES (100 mM) solution with water. The stock solutions of heparin (2.5 × 10−5 M and 2.5 × 10−4 M), the stock solutions of all anions and proteins (2.5 × 10−4 M) were prepared in 10 mM HEPES buffer (pH 7.4) solution and stored at 0–4 °C. The aqueous stock solution of IPy (2.5 × 10−4 M) was prepared in neat water. Biological samples were prepared using fetal bovine serum by diluting it with HEPES solution to the corresponding percentage in volume. All aqueous solutions were prepared from Milli-Q water (18.2 MΩ cm at 25 °C). 2.3. Heparin detection in aqueous buffer and serum solutions For detection in buffer solution, the testing solution containing IPy (1, 5, 10, 20, or 50 μM) was first prepared by diluting the aqueous stock solution of IPy with HEPES buffer solution (10 mM, pH 7.4). Then, 2.5 mL of testing solution was put in a quartz cell and the corresponding emission spectra were scanned. Next, heparin stock solution was added dropwise into the quartz cell and stirred by capillary tube. Then the emission spectra were scanned right away to record heparin-induced variation. The added volume of heparin stock solution is b4%, and for most experiments b0.4% of the volume of the testing solution. When tested in serum solution, the serum solution containing certain percentage of serum (0.2%, 5% and 10%) was first prepared by dilution of the serum into HEPES buffer solution (10 mM, pH 7.4), and then the testing solution containing IPy (10 μM) was prepared by adding the stock solution of IPy (100 μL, 2.5 × 10−4 M) into the corresponding serum solutions (2.4 mL). The fluorescence assay of sensing heparin was similar to that measured in buffer solution. 2.4. Protamine detection in aqueous buffer and serum solutions The fluorescence assay of detecting protamine was quite similar to that of detecting heparin. The big difference was the preparation of the testing solution. For detection in buffer solution, the testing solution
Scheme 1. Structures of IPy (a) and major unit of heparin (b) used in this study.
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Fig. 1. (a) Fluorescence emission of IPy (10 μM) upon titration of heparin (0–0.7 μM); inset: fluorescence emission of IPy (10 μM) upon titration of heparin above 0.7 μM; (b) fluorescence intensity ratio of excimer (511 nm) to monomer (385 nm), IE/IM, as a function of concentration of heparin; inset: the linear relationship between IE/IM and heparin concentration from 0 to 100 nM.
of IPy/heparin (10 μM/0.7 μM) was prepared by adding stock solution of IPy (100 μL, 2.5 × 10− 4 M) and heparin stock solution (7 μL, 2.5 × 10−4 M) to 2.4 mL of HEPES buffer solution. When tested in serum solutions, the testing solution was prepared by adding stock solution of IPy (100 μL, 2.5 × 10−4 M) and heparin (7 μL, 2.5 × 10−4 M) to 2.4 mL HEPES buffer solutions containing 0.2 or 2% serum. 3. Results and discussion 3.1. Sensing behaviors of IPy to heparin The cationic mono-pyrene-based fluorophore, IPy, displays stable monomer emission in aqueous solution with a quantum yield of 0.392 (using quinolone sulfate in 0.1 M H2SO4 aqueous solution as reference). The absence of excimer emission indicates that the probe is monomolecularly dispersed in water phase. This could be attributed to its cationic charges that endow it with good solubility in water. The gradual titration of heparin induces significant emission changes of the probe. As shown in Fig. 1a, the monomer emission is remarkably reduced to ca. 6% of its original intensity along with gradually enhanced excimer emission upon increasing addition of heparin from 0.01 to 0.7 μM, where the concentration of IPy is controlled at 10 μM. Although the formed excimer exhibits a much smaller intensity compared to the original monomer, the color of the sensor solution is obviously changed from dark blue to bright green (inset of Fig. 1a). The isoemissive point
at 476 nm indicates the formation of excimer is at the expense of monomer. Thus, the present cationic probe may function as a ratiometric sensor for heparin. We also checked the ratiometric responses of IPy at different concentration to heparin, and found that the concentration of IPy significantly influences its performance of sensing heparin. As shown in Fig. S1 (Supporting Information, SI), low concentration of IPy (e.g., 1 and 5 μM) shows much smaller ratiometric responses to heparin, whereas, high concentration of IPy (e.g., 20 and 50 μM) exhibits poor sensitivity to low concentration of heparin although it provides quite larger ratiometric signals to high concentration of heparin. Therefore, 10 μM was selected as the optimized concentration for IPy as the sensor to detect heparin. The ratiometric responses of IPy were further analyzed to evaluate its quantitative sensing ability and sensitivity to heparin. As displayed in Fig. 1b, the intensity ratio of excimer at 511 nm to monomer at 385 nm (IE/IM) is gradually enhanced from 0.006 to 4.846 as increasing heparin concentration from 0 to 0.7 μM, which is ca. 800-fold enhancement. Moreover, a linear relationship between IE/IM and heparin concentration is observed over a low concentration range from 10 to 100 nM (inset of Fig. 1b). The corresponding linear regression equation is IE/IM = 0.005(±0.001) + 0.217(±0.009) [heparin] with the relative correlation coefficient of 0.991. The detection limit (DL) of IPy to heparin is determined to be ca. 8.5 nM (153 ng/mL) according to the 3σ IUPAC criteria, and the detailed determination process and
Fig. 2. Ratiometric responses of IPy (10 μM) to various anions (0.7 μM) in HEPES buffer solution; inset: photos of IPy solution (10 μM) upon adding 0.7 μM of different anions under the illumination of 365 nm UV light; (b) ratiometric responses of IPy (10 μM) to heparin (0.7 μM) in the presence of one equiv. of various anions (0.7 μM) in HEPES buffer solution.
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Scheme 2. Schematic representation of the process of IPy sensing heparin.
corresponding measurement figures are provided in Fig. S2 (SI). The DL of a few reported ratiometric sensors for heparin were ca. 20 nM [20], 30 nM [36], 0.157 μM [3], and 33 pM [21]. By comparison, the cationic IPy represents a very high sensitive sensor for heparin. Although it is not the highest one, the present sensor has advantages such as detection in total aqueous solution and fast response time (b1 min.). The response to heparin was detected right away after addition of heparin in the sensor solution following a few seconds of stirring by capillary tube, and no particular incubation time is needed.
shown in Fig. 2b, except Cho sulfate, all the other tested anions show slight influence on the ratiometric responses of IPy to heparin. Although the presence of Cho sulfate reduces the response extent to heparin, a much higher response to heparin is still observed than that without heparin, indicating heparin could be detected even in the presence of Cho sulfate. These results confirm the feasibility of IPy functioning as a ratiomertic sensor to heparin.
3.3. IPy-heparin binding studies 3.2. Selectivity of IPy sensing heparin To check the selectivity of IPy to heparin, the fluorescence responses of the sensor to different anions, including chondroitin sulfate (Cho sulfate), hyaluronic acid (Hya acid), bovine serum albumin (BSA), ATP, 3− CTP, UTP, SO2− 4 , PO4 , and citrate, were examined. The ratiometric responses of IPy to various anions are illustrated in Fig. 2a, where the added anions were controlled at 0.7 μM since this particular concentration of heparin was found to induce the largest fluorescence variation of the sensor solution. It turns out that all the measured anions except Cho sulfate could not induce remarkable ratiometric responses under the same condition. Exceptionally, Cho sulfate produces a similar but much smaller ratiometric response, where the decreased monomer emission intensity is still larger than the enhanced excimer emission (Fig. S3, SI). This selectivity to heparin is also evidenced by the color change of the sensor solution upon addition of 0.7 μM different anions (inset of Fig. 2a). Only heparin and Cho sulfate among the tested anions change the solution color from blue to green, where the former leads to bright green and the latter results in pale green. The selectivity to heparin was further measured in the presence of these possible interferents. For this purpose, the ratiometric responses of IPy to heparin (0.7 μM) were measured in the presence of one equiv. of each interferent. As
The ratiometric responses induced by heparin indicate that heparin draws pyrene moieties in close proximity to emit excimer emission. This could be due to the electrostatic interaction between the anionic macromolecule and the cationic probe that induces binding of IPy with heparin and leads to form heparin-IPy complex. As a result, the distance between pyrene moieties is shortened and helps to form excimer (Scheme 2). Such assumption is verified by UV–vis absorption and time-resolved decay measurements. The UV–vis absorption spectra of IPy upon titration of heparin are illustrated in Fig. 3a. Clearly, the probe itself exhibits mainly pyrenyl monomer bands at 280, 350 and 380 nm. The gradual addition of heparin leads to continuous reduction of the three absorption bands accompanied with an obvious spectral red shift. These results are attributed to the intermolecular π-π stacking of two pyrene moieties in the ground state [3,5], suggesting the intermolecular excimer among the cationic probes was formed from binding with anionic heparin. Differently, the addition of ATP leads to neglect absorption changes (Fig. S4, SI). This suggests that the selective ratiometric responses to heparin are attributed to the strong electrostatic interaction between heparin and IPy. The time-resolved decay of IPy upon titration of heparin is shown in Fig. 3b. The decay was monitored at 385 nm to see the changes of fluorescence lifetime of pyrene moieties for monomer emission. The
Fig. 3. (a) UV–vis absorption of IPy (10 μM) upon titration of heparin in HEPES buffer solution (10 mM, pH 7.4); (b) time-resolved fluorescence decay of IPy (10 μM) upon titration of heparin in HEPES buffer solution (10 mM, pH 7.4), inset: fluorescence decay lifetimes of IPy upon titration of heparin.
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Fig. 4. (a) Fluorescence emission spectra of IPy/heparin (10 μM/0.7 μM) system upon titration of protamine in HEPES buffer (10 mM, pH 7.4); (b) fluorescence intensity ratio of monomer to excimer (IM/IE) as a function of the concentration of protamine; inset: the quadratic relationship between IM/IE and protamine concentration from 0.1 to 0.7 μM.
corresponding decay times were obtained from the multiexponential fitting of the decays according to Eq. (1) and listed in the inset of Fig. 3b. Rðt Þ ¼ B1 eð−t=τ1 Þ þ B2 eð−t=τ2 Þ
ð1Þ
It can be seen that the probe shows only one lifetime in the absence of heparin, which is ca. 6.8 ns and can be ascribed to the lifetime of the free probe. The addition of heparin leads to existence of two lifetimes of pyrene moieties. A new longer lifetime (ca. 10.3 ns) appears and gradually increases to 21.9 ns along increasing heparin concentration. This new one can be ascribed to the lifetime of the probe binding with heparin, verifying the existence of two different binding states of IPy. The binding constant value of IPy and heparin was determined from the emission intensity data at 385 nm following the modified BenesiHildebrand equation, 1/Δ I = 1/Δ Imax + (1/K[C]) (1/Δ Imax) [37]. For the present system, Δ I = Imax − I, Δ Imax = Imax − Imin, where Imax, I and Imin are the emission intensity of IPy at 385 nm in the absence of heparin, in the presence of heparin at an intermediate concentration, and in the presence of 0.7 μM heparin, respectively, K is the binding constant, and [C] is heparin concentration. The plot of (Imax − Imin)/(Imax − I) against [heparin]−1 is shown in Fig. S5 (SI), and the value of K was determined to be 3.9 × 106 M−1 from the slope. This value reveals the cationic probe exhibits a quite high heparin-binding affinity, which endows it with high sensitivity and selectivity to heparin. It is to be noted that a turning point appears at 0.7 μM of heparin, after which the further addition of heparin induces rise-up of monomer emission, slight reduction of excimer emission (inset of Fig. 1a), and
decrease of the ratio of excimer to monomer, IE/IM (Fig. 1b). This may be due to the binding between IPy and heparin has reached their maximum combining degree. Continuously added free heparin competes with IPy-binding heparin to complex with the cationic probe, which reduces the number of pyrene derivatives on heparin and leads to far distance among pyrene moieties and a resulting enhanced monomer emission (Scheme 2). UV–vis measurements reveal that above 0.7 μM, further titration of heparin into solution of IPy causes enhancement of the three absorption bands and spectral blue-shift, verifying the break-up of π-π complex between pyrene moieties (Fig. S6, SI). This indicates that the binding between the cationic probe and anionic heparin is dynamic and reversible, which makes IPy/heparin complex a possible ratiometric sensor for analytes having stronger heparin-binding affinity. 3.4. IPy/heparin complex as sensing platform for protamine It is known that protamine has strong heparin-binding affinity and protamine-heparin binding constants are in the range of 5.4– 212 × 106 M−1 for large molecular weight heparin [38]. The larger binding constant can enable protamine to compete with the cationic IPy to bind with heparin and free the probe from the complex. As a result, the probe is released back to aqueous solution and emits monomer emission. If this is the case, the IPy/heparin complex can function as a green-to-blue ratiometric sensor for protamine. Therefore, the IPy/heparin complex containing 10 μM of probe and 0.7 μM of heparin was selected as the sensor platform since it emits the largest intensity ratio of excimer to monomer. Fig. 4a illustrates the fluorescence variation of
Fig. 5. Ratiometric responses of IPy/heparin (10 μM/0.7 μM) to various proteins and amino acids (2.5 μM). Inset: photos of IPy/heparin (10 μM/0.7 μM) solution upon adding 2.5 μM of different proteins under the illumination of 365 nm UV light; (b) ratiometric responses of IPy/heparin (10 μM/0.7 μM) to protamine (2.5 μM) in the presence of one equiv. of various proteins and amino acids. Inset: the enlarged ratiometric responses of the binary system to protamine (2.5 μM) in the presence of one equiv. of TPS and pepsin.
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Fig. 6. (a) UV–vis absorption of IPy/heparin (10 μM/0.7 μM) upon titration of protamine in HEPES buffer solution (10 mM, pH 7.4); (b) time-resolved decays of IPy/heparin (10 μM/0.7 μM) upon titration of protamine in HEPES buffer solution (10 mM, pH 7.4), inset: fluorescence decay lifetime data of IPy/heparin upon titration of protamine.
IPy/heparin upon titration of protamine. Obviously, it shows reversed ratiometric responses compared to IPy sensing heparin. The monomer intensity is significantly enhanced, at the same time the excimer emission is gradually reduced. An isoemissive point is observed at 471 nm. Clearly, this binary system also provides a ratiometric response to protamine. The green solution of the binary system changes back to blue upon addition of protamine (inset of Fig. 4a). The intensity ratio of monomer to excimer, IM/IE, is firstly gradually increased from 0.28 to 3.55 upon addition of protamine from 0 to 1.0 μM, and then abruptly and significantly increased to 165.10 upon further addition of protamine to 2.5 μM, which is ca. 600-fold enhancement. The further addition of protamine above 2.5 μM leads to no further enhancement of IM/IE, indicating the binding of protamine with heparin reaches saturation. Moreover, the ratiometric signal, IM/IE, of this binary sensor exhibits a good quadratic relationship with protamine concentration below 0.7 μM (inset of Fig. 4b). The corresponding equation is expressed as IM/IE = 0.280 (± 0.009) + 0.460 (± 0.071) [protamine] + 0.622 (± 0.134) [protamine]2 with the relative correlation coefficient of 0.997. Similar ratiometric response behaviors were also found for IPy/ heparin systems with different concentration of heparin (Fig. S7, SI). The DLs of these binary sensor systems were determined according to the standard 3σ criteria using their ratiometric responsive data (Fig. S8, SI), which are 15.4 nM (107.8 ng/mL), 25.8 nM (180.6 ng/mL), 27.2 nM (190.4 ng/mL), 77.6 nM (543.2 ng/mL), and 83.7 nM (585.9 ng/mL) for the binary systems containing heparin at 0.4, 0.5, 0.7, 0.8, and 1.0 μM, respectively. Clearly, low heparin concentration endows the binary system with high sensitivity, but leads to narrow responsive range. Although the sensitivity is not quite high compared to some reported sensors for protamine [8,19,26], the present sensor is, to the best of our knowledge, the only ratiometric sensor for protamine. Moreover, the response to protamine is fast and b1 min. 3.5. Selectivity of IPy/heparin to protamine To examine the selectivity of the binary system to protamine, the fluorescence response of IPy/heparin to different proteins and amino acids was measured. The examined proteins include poly-lysine, HIV-1 TAT, lysozyme (LYZ), trypsin (TPS), β-lactoglobulin (BLG), BSA, ovalbumin egg (O-egg), pepsin, and cytochrome c (Cyt-c). The measured positively charged amino acids include arginine (Arg), lysine (Lys) and histidine (His). As shown in Fig. 5a, among all the tested proteins at 2.5 μM, only protamine could produce prominent increases of the intensity ratio of IM/IE. Poly-lysine could also induce some ratiometric responses at this concentration, which is much smaller than that by protamine. TAT containing several Arg residues, and small positively charged amino acids (Arg, Lys, and His) could not produce apparent ratiometric responses. These results suggest that the large IPy-heparin
binding constant endow it with high selectivity to protamine that has even higher heparin-binding affinity. The high selectivity of the binary sensor system to protamine is also verified by the visual color change. As shown in the inset of Fig. 5a, only protamine and poly-lysine could change the green solution back to blue when exposed to 365 nm UV light, where the former leads to bright blue and the latter induces pale blue. The selectivity to protamine was further measured in the presence of these possible interferents. To this end, the ratiometric responses of the binary sensor were measured in the presence of one equiv. of each interferent. As shown in Fig. 5b, all the tested proteins or amino acids (except TPS and pepsin) display faint influence on the ratiometric responses to protamine. The presence of TPS and pepsin decreases the response extent to protamine. Even though, N10 times higher response to protamine is still observed than that without protamine (inset of Fig. 5b), indicating protamine could be detected even in the presence of TPS or pepsin. These results verify the feasibility of IPy/heparin binary sensor functioning as a ratiomertic sensor to protamine. 3.6. Sensing mechanism of IPy/heparin detecting protamine The process of IPy/heparin sensing protamine was also examined by UV–vis absorption and time-resolved decay measurements. As shown in Fig. 6a, along increasing protamine concentration, the blunt absorption bands of the binary system at 280, 350 and 380 nm gradually increase and become sharp accompanied with an obvious blue-shift, suggesting break-up of π-π stacking between pyrene moieties and giving rise to existence as monomer again. This reveals, to a certain extent, that heparin prefers to binding protamine to the probe due to the stronger affinity between protamine and heparin. As a result, the former electrostatic balance between the cationic probe and anionic heparin was broken, which leads to release of the probe from the anionic macromolecules (Scheme 3). As a control, the addition of lysozyme does not induce similar changes of UV–vis absorption of IPy/heparin (Fig. S9, SI), suggesting the electrostatic interaction between heparin and protamine plays an important role in the selective responses to protamine.
Scheme 3. Schematic representation of the process of IPy/heparin sensing protamine.
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Fig. 7. (a) Fluorescence emission of IPy (10 μM) in the absence and presence of heparin (0.3 and 2.0 μM); (b) color change of IPy solutions (10 μM) containing 0.3 and 2.0 μM heparin upon addition of different amount of protamine.
The time-resolved fluorescence decays of IPy/heparin upon titration of protamine are displayed in Fig. 6b, which were also monitored at 385 nm. The corresponding decay lifetimes are listed in the inset of Fig. 6b. It can be seen, along increasing protamine concentration, the two lifetimes of pyrene moieties change back to only one lifetime, suggesting the binding-state pyrene moieties change to non-binding state. Such results verify that the addition of protamine induces release of cationic probe from binding with heparin and gives rise to monomer emission, which undoubtedly was due to the stronger binding affinity of protamine with heparin.
free the probe from the complex with heparin and realize the blue color of the solution. To check the possibility of this methodology, we added protamine into the above two solutions and monitored the color change. As shown in Fig. 7b, the two solutions containing 0.3 μM and 2.0 μM heparin separately exhibit similar green color. The former needs 1.6 μM protamine to realize the solution color change back to blue, however, the latter needs ca. 10 μM protamine to accomplish the color change. Therefore, a proper differentiation between low concentrated heparin and high concentrated one could be realized by the subsequent added protamine.
3.7. Differentiation of heparin at low and high concentration
3.8. Ratiometric detection of heparin and protamine in serum solutions
As revealed earlier in Fig. 1a, the addition of heparin larger than 0.7 μM reduces the ratiometric responses as a result of dilution of aggregated pyrene probes with free heparins. Thus, a determined value of IE/ IM could be related with two different heparin concentrations: one is smaller than 0.7 μM and the other is larger than 0.7 μM. For example, as shown in Fig. 7a, the fluorescence emission of the probe (10 μM) changes to similar profile upon addition of 0.3 μM and 2.0 μM heparin. Moreover, the observed ratio of excimer to monomer is also similar (inset of Fig. 7a). Therefore, it is necessary to differentiate heparin at high concentration from that at low concentration. Considering the color change of IPy/heparin induced by protamine, it is possible to recognize the real heparin concentration by the amount of protamine used to reverse the solution color back to blue. This is because the binary system with high concentration of heparin should need more protamine to
Considering the important values of the potential application in detecting biological sample, we then measured the fluorescence responses of IPy to heparin and that of IPy/heparin system to protamine in bovine serum solution. As shown in Fig. 8a, the ratiometric response to heparin is also observed for IPy in aqueous buffer solution containing 0.2% bovine serum. Approximately 80-fold enhancement of IE/IM was obtained from 0.019 to 1.546 by addition of 1.0 μM heparin (Fig. 8b). We also measured the fluorescence responses of IPy to heparin in more concentrated serum solutions, such as 5% and 10% bovine serum. Similar ratiometric responses were observed again, where the monomer emission was decreased accompanied by the increased excimer emission upon continuous addition of heparin from 0 to 2 μM (Fig. S10, SI). It is just that the extent of the ratiometric responses is reduced in more concentrated serum solutions. Even though, a linear relationship still exists
Fig. 8. (a) Fluorescence spectra of IPy (10 μM) upon titration of heparin from 0 to 1.0 μM in 0.2% bovine serum solution; inset: fluorescence spectra of IPy (10 μM) upon titration of heparin from 1.0 to 10 μM in 0.2% bovine serum solution; (b) fluorescence intensity ratio of excimer to monomer (IE/IM) of IPy (10 μM) as a function of heparin concentration measured in 0.2% bovine serum solution.
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between the ratiometric response (IE/IM) and heparin concentration. We then checked the detection of protamine by the binary system based on IPy/heparin in biological solutions containing 0.2% and 2% bovine serum. Again, as in aqueous solution, a similar reversed ratiometric response expressed as decreased excimer and enhanced monomer was observed in serum solutions (Fig. S11 and S12, SI). Moreover, the ratiometric responses (IM/IE) of the binary system show linear relationship with protamine concentration below 0.8 μM in 2% serum solutions (inset of Fig. S12b). Such results suggest that applying the detection process of both IPy sensing heparin and IPy/heparin sensing protamine in biological samples is quite promising. 4. Conclusion The cationic imidazolium-modified pyrene derivative, IPy, can be used to function as a ratiometric fluorescent sensor for detection of heparin based on variation of monomer and excimer emission. The probe selectively exhibits remarkable monomer reduction along with excimer enhancement to the presence of heparin in HEPES buffer solution. The cationic probe has large heparin-binding affinity with binding constant of 3.9 × 106 M−1. In addition, the binary combination of IPy/heparin can realize highly sensitive and selective ratiometric detection of protamine among the tested proteins and positively charged amino acids. The larger heparin-binding affinity enables protamine to replace the heparinbound IPy and release the probe back to non-binding state, leading to enhanced monomer emission along decreased excimer emission. The detection limits of heparin and protamine were measured to be as low as 8.5 (153 ng/mL) and 15.4 nM (107.8 ng/mL), respectively. Moreover, the proposed method could be used for ratiometric detection of both heparin and protamine in biological samples. Acknowledgments The authors acknowledge the financial support from National Natural Science Foundation of China (21573140), Ministry of Education of China (NCET-12-0895), the Program of Introducing Talents of Discipline to Universities (B14041), the Program for Changjiang Scholars and Innovative Research Team in University (IRT_14R33). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.saa.2016.07.026.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
A pyrene-based fluorescent sensor for ratiometric detection of heparin and its complex with heparin for reversed ratiometric detection of protamine in aqueous solution Weiwei Gong, Shihuai Wang, Yuting Wei, Liping Ding ⁎, Yu Fang Key Laboratory of Applied Surface and Colloid Chemistry of Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, PR China
a r t i c l e
i n f o
Article history: Received 20 April 2016 Received in revised form 13 July 2016 Accepted 14 July 2016 Available online 16 July 2016 Keywords: Heparin Protamine Excimer Fluorescence Serum solution
a b s t r a c t An imidazolium-modified pyrene derivative, IPy, was used for ratiometric detection of heparin, and its complex with heparin was used for reversed ratiometric detection of protamine in both aqueous solution and serum samples. The cationic fluorescent probe could interact with anionic heparin via electrostatic interaction to bring about blue-to-green fluorescence changes as monomer emission significantly decreases and excimer increases. The binary combination of IPy and heparin could be further used for green-to-blue detection of protamine since heparin prefers to bind to protamine instead of the probe due to its stronger affinity with protamine. The cationic probe shows high sensitivity to heparin with a low detection limit of 8.5 nM (153 ng/mL) and its combination with heparin displays high sensitivity to protamine with a detection limit as low as 15.4 nM (107.8 ng/mL) according to the 3σ IUPAC criteria. Moreover, both sensing processes are fast and can be performed in serum solutions, indicating possibility for practical applications. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Heparin is a linear polysaccharide with abundant of sulfate and carboxylate groups, and therefore has the highest negative charge density among any known biological macromolecules [1,2]. Heparin plays an important role in regulating various biological processes, such as cell growth and differentiation, inflammation, and blood coagulation [3,4]. More importantly, heparin has been widely used in clinical applications such as preventing thrombosis during surgery and as an anticoagulant drug to treat thrombotic diseases [5]. However, overdose of heparin can induce some complications such as hemorrhages and heparin-induced thrombocytopenia [3]. The suggested therapeutic dose of heparin is 2–8 U/mL (17–67 μM) during cardiovascular surgery and 0.2–1.2 U/mL (1.7–10 μM) for postoperative and long-term care, respectively [6,7]. Protamine, as a well-known heparin antidote, is a highly cationic protein (pI = 13.8) with high content of basic arginine residues. The anticoagulant effect of heparin can be reversed by protamine due to their combination through electrostatic interaction [8]. Thus, protamine is often used for treatment of heparin overdose. However, the over-use of protamine could also induce adverse effect such as hypotension and idiosyncratic fatal cardiac arrest [9]. Therefore, the detection and quantification of both heparin and protamine are of crucial significance for clinical procedures.
⁎ Corresponding author. E-mail address: [email protected] (L. Ding).
http://dx.doi.org/10.1016/j.saa.2016.07.026 1386-1425/© 2016 Elsevier B.V. All rights reserved.
Among various methods including colorimetric sensor [10,11], electrochemical sensor [4], and surface-enhanced Raman sensor [12], fluorescent sensors have drawn extensive attention because of their advantages in terms of high sensitivity, high selectivity, easy operation and real time detection [13]. Up to now, a great amount of fluorescent sensors have been developed for detecting heparin and protamine. These sensors exhibit either turn-off responses [2,14–16] or turn-on [17–19] or ratiometric responses [3,5,20,21] to the presence of the target analyte. The turn-off and turn-on sensors rely on fluorescence intensity variation at one wavelength and are easily affected by instrumental and environmental conditions. By comparison, ratiometric fluorescent sensors are advantageous because they usually depend on the intensity ratio at two emission wavelengths, which can help avoiding the abovementioned interferences [22]. Most of the above mentioned fluorescent sensors detect only one target, either heparin or protamine. Very recently, fluorescent sensors for recognizing both heparin and protamine are attracting attention since they are more convenient and multi-functional. Zhang and coworkers used gold nanoparticles coupled with fluorescent emitting materials such as silicon quantum dots [23], fluorescein [24], and upconversion nanoparticles [25] for detecting both protamine and heparin. These gold nanoparticle-attached fluorescent materials are fluorescence inert due to the quenching effect of gold surface, and show turn-on responses to protamine as protamine interacts with gold nanoparticles and de-attaches fluorescent materials from gold surface, and then show turn-off responses to heparin because heparin has stronger affinity with protamine that results in binding fluorescent materials
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with gold nanoparticles again. John et al. reported using folic acid capped gold nanoparticles for detecting both protamine and heparin based on aggregation and de-aggregation of gold nanoparticles induced by the added analyte [26]. Su et al. developed a fluorescein-labeled DNA probe for turn-off sensing protamine and turn-on sensing heparin [8]. Although these sensors show some interesting sensing behaviors, they have some drawbacks like long time of incubation [23–25], or low sensitivity [9]. Moreover, they are all based on single signal of fluorescence variation at one wavelength. Fluorescent ratiometric sensors for detecting both heparin and protamine have never been reported, to our knowledge. In the present study, we reported using a cationic pyrene-based fluorophore for ratiometric detection of both heparin and protamine in total aqueous solution. Pyrene-based fluorophores have been widely used to develop ratiometric sensors as they can emit monomer and excimer emission [27–29], and more recently used for ratiometric detection of various biomolecules such as G-quadruplex structures of Grich DNA [30], human breast cancer cell [31], and proteins [32]. Their fluorescence emission is highly dependent on the conformation or aggregation of pyrene derivatives and very sensitive to the microenvironment. Although a variety of pyrene derivatives were reported for ratiometric sensing heparin [3,5,21,33,34], they were not used to detect protamine or both targets. The present imidazolium-modified pyrene derivative (IPy, Scheme 1) exhibits only monomer emission in aqueous buffer solution, and shows blue-to-green emission change upon addition of heparin as monomer emission significantly decreases accompanied by excimer emission increasing. The binary combination of IPy and heparin displays green-to-blue changes to the addition of protamine as witnessed by recovered monomer emission and decreased excimer emission. Moreover, both sensing processes show high sensitivity and can be performed in serum solutions.
2. Experimental methods 2.1. Materials and instruments The pyrene derivative (IPy) was synthesized and characterized according to our previously reported method [35]. Heparin sodium salt (from hog intestine) was purchased from TCI. N-(2-hydroxyethyl) piperazine-Nʹ-ethanesulfonic acid (HEPES), chondroitin sulfate sodium salt, adenosine 5ʹ-triphosphate disodium salt hydrate (ATP, ≥99%), cytidine 5ʹ-triphosphate disodium salt (CTP, ≥95%), uridine 5ʹ-triphosphate trisodium salt hydrate (UTP, ≥96%), L-arginine (99%), L-lysine (99%), Lhistidine (99%) and all proteins including protamine (from salmon), bovine serum albumin (≥ 98%), lysozyme (from chicken egg, ≥ 90%), trypsin (from bovine pancreas), pepsin (from porcine gastric mucosa), β-lactoglobulin (from bovine milk, ≥90%), cytochrome c (from bovine heart, ≥95%), ovalbumin egg (from chicken egg white, ≥ll p, hyaluronic acid sodium salt, and poly-L-lysine hydrobromide were purchased from Sigma-Aldrich. HIV-1 TAT peptide (Sequence: YGRKKRRQRRR) was obtained from Chinese Peptide Company. Fetal bovine serum was
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purchased from Zhejiang Tianhang Biotechnology Co. Ltd. All chemicals were used as received. Steady-state fluorescence spectra were measured on a single photon-counting fluorescence spectrometer (FS5, Edinburgh Instruments) with xenon light (150 W) as the excitation source, and the excitation and emission slit widths were set at 2.1 and 0.3 nm, respectively. All samples were excited at 346 nm. Time-resolved fluorescence decays were measured on a time-resolved single photon-counting fluorescence spectrometer (FS920, Edinburgh Instruments) with 343.4 nm laser as the excitation source. UV–vis absorption spectra were recorded on a spectrophotometer (U3900, Hitachi Instrument). 2.2. Preparation of samples The aqueous stock solution of HEPES (100 mM) was first prepared by dissolving solid HEPES in neat water and using NaOH (1.0 M) to regulate pH to 7.4. HEPES buffer solution (10 mM, pH 7.4) was prepared by diluting the stock HEPES (100 mM) solution with water. The stock solutions of heparin (2.5 × 10−5 M and 2.5 × 10−4 M), the stock solutions of all anions and proteins (2.5 × 10−4 M) were prepared in 10 mM HEPES buffer (pH 7.4) solution and stored at 0–4 °C. The aqueous stock solution of IPy (2.5 × 10−4 M) was prepared in neat water. Biological samples were prepared using fetal bovine serum by diluting it with HEPES solution to the corresponding percentage in volume. All aqueous solutions were prepared from Milli-Q water (18.2 MΩ cm at 25 °C). 2.3. Heparin detection in aqueous buffer and serum solutions For detection in buffer solution, the testing solution containing IPy (1, 5, 10, 20, or 50 μM) was first prepared by diluting the aqueous stock solution of IPy with HEPES buffer solution (10 mM, pH 7.4). Then, 2.5 mL of testing solution was put in a quartz cell and the corresponding emission spectra were scanned. Next, heparin stock solution was added dropwise into the quartz cell and stirred by capillary tube. Then the emission spectra were scanned right away to record heparin-induced variation. The added volume of heparin stock solution is b4%, and for most experiments b0.4% of the volume of the testing solution. When tested in serum solution, the serum solution containing certain percentage of serum (0.2%, 5% and 10%) was first prepared by dilution of the serum into HEPES buffer solution (10 mM, pH 7.4), and then the testing solution containing IPy (10 μM) was prepared by adding the stock solution of IPy (100 μL, 2.5 × 10−4 M) into the corresponding serum solutions (2.4 mL). The fluorescence assay of sensing heparin was similar to that measured in buffer solution. 2.4. Protamine detection in aqueous buffer and serum solutions The fluorescence assay of detecting protamine was quite similar to that of detecting heparin. The big difference was the preparation of the testing solution. For detection in buffer solution, the testing solution
Scheme 1. Structures of IPy (a) and major unit of heparin (b) used in this study.
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Fig. 1. (a) Fluorescence emission of IPy (10 μM) upon titration of heparin (0–0.7 μM); inset: fluorescence emission of IPy (10 μM) upon titration of heparin above 0.7 μM; (b) fluorescence intensity ratio of excimer (511 nm) to monomer (385 nm), IE/IM, as a function of concentration of heparin; inset: the linear relationship between IE/IM and heparin concentration from 0 to 100 nM.
of IPy/heparin (10 μM/0.7 μM) was prepared by adding stock solution of IPy (100 μL, 2.5 × 10− 4 M) and heparin stock solution (7 μL, 2.5 × 10−4 M) to 2.4 mL of HEPES buffer solution. When tested in serum solutions, the testing solution was prepared by adding stock solution of IPy (100 μL, 2.5 × 10−4 M) and heparin (7 μL, 2.5 × 10−4 M) to 2.4 mL HEPES buffer solutions containing 0.2 or 2% serum. 3. Results and discussion 3.1. Sensing behaviors of IPy to heparin The cationic mono-pyrene-based fluorophore, IPy, displays stable monomer emission in aqueous solution with a quantum yield of 0.392 (using quinolone sulfate in 0.1 M H2SO4 aqueous solution as reference). The absence of excimer emission indicates that the probe is monomolecularly dispersed in water phase. This could be attributed to its cationic charges that endow it with good solubility in water. The gradual titration of heparin induces significant emission changes of the probe. As shown in Fig. 1a, the monomer emission is remarkably reduced to ca. 6% of its original intensity along with gradually enhanced excimer emission upon increasing addition of heparin from 0.01 to 0.7 μM, where the concentration of IPy is controlled at 10 μM. Although the formed excimer exhibits a much smaller intensity compared to the original monomer, the color of the sensor solution is obviously changed from dark blue to bright green (inset of Fig. 1a). The isoemissive point
at 476 nm indicates the formation of excimer is at the expense of monomer. Thus, the present cationic probe may function as a ratiometric sensor for heparin. We also checked the ratiometric responses of IPy at different concentration to heparin, and found that the concentration of IPy significantly influences its performance of sensing heparin. As shown in Fig. S1 (Supporting Information, SI), low concentration of IPy (e.g., 1 and 5 μM) shows much smaller ratiometric responses to heparin, whereas, high concentration of IPy (e.g., 20 and 50 μM) exhibits poor sensitivity to low concentration of heparin although it provides quite larger ratiometric signals to high concentration of heparin. Therefore, 10 μM was selected as the optimized concentration for IPy as the sensor to detect heparin. The ratiometric responses of IPy were further analyzed to evaluate its quantitative sensing ability and sensitivity to heparin. As displayed in Fig. 1b, the intensity ratio of excimer at 511 nm to monomer at 385 nm (IE/IM) is gradually enhanced from 0.006 to 4.846 as increasing heparin concentration from 0 to 0.7 μM, which is ca. 800-fold enhancement. Moreover, a linear relationship between IE/IM and heparin concentration is observed over a low concentration range from 10 to 100 nM (inset of Fig. 1b). The corresponding linear regression equation is IE/IM = 0.005(±0.001) + 0.217(±0.009) [heparin] with the relative correlation coefficient of 0.991. The detection limit (DL) of IPy to heparin is determined to be ca. 8.5 nM (153 ng/mL) according to the 3σ IUPAC criteria, and the detailed determination process and
Fig. 2. Ratiometric responses of IPy (10 μM) to various anions (0.7 μM) in HEPES buffer solution; inset: photos of IPy solution (10 μM) upon adding 0.7 μM of different anions under the illumination of 365 nm UV light; (b) ratiometric responses of IPy (10 μM) to heparin (0.7 μM) in the presence of one equiv. of various anions (0.7 μM) in HEPES buffer solution.
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Scheme 2. Schematic representation of the process of IPy sensing heparin.
corresponding measurement figures are provided in Fig. S2 (SI). The DL of a few reported ratiometric sensors for heparin were ca. 20 nM [20], 30 nM [36], 0.157 μM [3], and 33 pM [21]. By comparison, the cationic IPy represents a very high sensitive sensor for heparin. Although it is not the highest one, the present sensor has advantages such as detection in total aqueous solution and fast response time (b1 min.). The response to heparin was detected right away after addition of heparin in the sensor solution following a few seconds of stirring by capillary tube, and no particular incubation time is needed.
shown in Fig. 2b, except Cho sulfate, all the other tested anions show slight influence on the ratiometric responses of IPy to heparin. Although the presence of Cho sulfate reduces the response extent to heparin, a much higher response to heparin is still observed than that without heparin, indicating heparin could be detected even in the presence of Cho sulfate. These results confirm the feasibility of IPy functioning as a ratiomertic sensor to heparin.
3.3. IPy-heparin binding studies 3.2. Selectivity of IPy sensing heparin To check the selectivity of IPy to heparin, the fluorescence responses of the sensor to different anions, including chondroitin sulfate (Cho sulfate), hyaluronic acid (Hya acid), bovine serum albumin (BSA), ATP, 3− CTP, UTP, SO2− 4 , PO4 , and citrate, were examined. The ratiometric responses of IPy to various anions are illustrated in Fig. 2a, where the added anions were controlled at 0.7 μM since this particular concentration of heparin was found to induce the largest fluorescence variation of the sensor solution. It turns out that all the measured anions except Cho sulfate could not induce remarkable ratiometric responses under the same condition. Exceptionally, Cho sulfate produces a similar but much smaller ratiometric response, where the decreased monomer emission intensity is still larger than the enhanced excimer emission (Fig. S3, SI). This selectivity to heparin is also evidenced by the color change of the sensor solution upon addition of 0.7 μM different anions (inset of Fig. 2a). Only heparin and Cho sulfate among the tested anions change the solution color from blue to green, where the former leads to bright green and the latter results in pale green. The selectivity to heparin was further measured in the presence of these possible interferents. For this purpose, the ratiometric responses of IPy to heparin (0.7 μM) were measured in the presence of one equiv. of each interferent. As
The ratiometric responses induced by heparin indicate that heparin draws pyrene moieties in close proximity to emit excimer emission. This could be due to the electrostatic interaction between the anionic macromolecule and the cationic probe that induces binding of IPy with heparin and leads to form heparin-IPy complex. As a result, the distance between pyrene moieties is shortened and helps to form excimer (Scheme 2). Such assumption is verified by UV–vis absorption and time-resolved decay measurements. The UV–vis absorption spectra of IPy upon titration of heparin are illustrated in Fig. 3a. Clearly, the probe itself exhibits mainly pyrenyl monomer bands at 280, 350 and 380 nm. The gradual addition of heparin leads to continuous reduction of the three absorption bands accompanied with an obvious spectral red shift. These results are attributed to the intermolecular π-π stacking of two pyrene moieties in the ground state [3,5], suggesting the intermolecular excimer among the cationic probes was formed from binding with anionic heparin. Differently, the addition of ATP leads to neglect absorption changes (Fig. S4, SI). This suggests that the selective ratiometric responses to heparin are attributed to the strong electrostatic interaction between heparin and IPy. The time-resolved decay of IPy upon titration of heparin is shown in Fig. 3b. The decay was monitored at 385 nm to see the changes of fluorescence lifetime of pyrene moieties for monomer emission. The
Fig. 3. (a) UV–vis absorption of IPy (10 μM) upon titration of heparin in HEPES buffer solution (10 mM, pH 7.4); (b) time-resolved fluorescence decay of IPy (10 μM) upon titration of heparin in HEPES buffer solution (10 mM, pH 7.4), inset: fluorescence decay lifetimes of IPy upon titration of heparin.
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Fig. 4. (a) Fluorescence emission spectra of IPy/heparin (10 μM/0.7 μM) system upon titration of protamine in HEPES buffer (10 mM, pH 7.4); (b) fluorescence intensity ratio of monomer to excimer (IM/IE) as a function of the concentration of protamine; inset: the quadratic relationship between IM/IE and protamine concentration from 0.1 to 0.7 μM.
corresponding decay times were obtained from the multiexponential fitting of the decays according to Eq. (1) and listed in the inset of Fig. 3b. Rðt Þ ¼ B1 eð−t=τ1 Þ þ B2 eð−t=τ2 Þ
ð1Þ
It can be seen that the probe shows only one lifetime in the absence of heparin, which is ca. 6.8 ns and can be ascribed to the lifetime of the free probe. The addition of heparin leads to existence of two lifetimes of pyrene moieties. A new longer lifetime (ca. 10.3 ns) appears and gradually increases to 21.9 ns along increasing heparin concentration. This new one can be ascribed to the lifetime of the probe binding with heparin, verifying the existence of two different binding states of IPy. The binding constant value of IPy and heparin was determined from the emission intensity data at 385 nm following the modified BenesiHildebrand equation, 1/Δ I = 1/Δ Imax + (1/K[C]) (1/Δ Imax) [37]. For the present system, Δ I = Imax − I, Δ Imax = Imax − Imin, where Imax, I and Imin are the emission intensity of IPy at 385 nm in the absence of heparin, in the presence of heparin at an intermediate concentration, and in the presence of 0.7 μM heparin, respectively, K is the binding constant, and [C] is heparin concentration. The plot of (Imax − Imin)/(Imax − I) against [heparin]−1 is shown in Fig. S5 (SI), and the value of K was determined to be 3.9 × 106 M−1 from the slope. This value reveals the cationic probe exhibits a quite high heparin-binding affinity, which endows it with high sensitivity and selectivity to heparin. It is to be noted that a turning point appears at 0.7 μM of heparin, after which the further addition of heparin induces rise-up of monomer emission, slight reduction of excimer emission (inset of Fig. 1a), and
decrease of the ratio of excimer to monomer, IE/IM (Fig. 1b). This may be due to the binding between IPy and heparin has reached their maximum combining degree. Continuously added free heparin competes with IPy-binding heparin to complex with the cationic probe, which reduces the number of pyrene derivatives on heparin and leads to far distance among pyrene moieties and a resulting enhanced monomer emission (Scheme 2). UV–vis measurements reveal that above 0.7 μM, further titration of heparin into solution of IPy causes enhancement of the three absorption bands and spectral blue-shift, verifying the break-up of π-π complex between pyrene moieties (Fig. S6, SI). This indicates that the binding between the cationic probe and anionic heparin is dynamic and reversible, which makes IPy/heparin complex a possible ratiometric sensor for analytes having stronger heparin-binding affinity. 3.4. IPy/heparin complex as sensing platform for protamine It is known that protamine has strong heparin-binding affinity and protamine-heparin binding constants are in the range of 5.4– 212 × 106 M−1 for large molecular weight heparin [38]. The larger binding constant can enable protamine to compete with the cationic IPy to bind with heparin and free the probe from the complex. As a result, the probe is released back to aqueous solution and emits monomer emission. If this is the case, the IPy/heparin complex can function as a green-to-blue ratiometric sensor for protamine. Therefore, the IPy/heparin complex containing 10 μM of probe and 0.7 μM of heparin was selected as the sensor platform since it emits the largest intensity ratio of excimer to monomer. Fig. 4a illustrates the fluorescence variation of
Fig. 5. Ratiometric responses of IPy/heparin (10 μM/0.7 μM) to various proteins and amino acids (2.5 μM). Inset: photos of IPy/heparin (10 μM/0.7 μM) solution upon adding 2.5 μM of different proteins under the illumination of 365 nm UV light; (b) ratiometric responses of IPy/heparin (10 μM/0.7 μM) to protamine (2.5 μM) in the presence of one equiv. of various proteins and amino acids. Inset: the enlarged ratiometric responses of the binary system to protamine (2.5 μM) in the presence of one equiv. of TPS and pepsin.
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Fig. 6. (a) UV–vis absorption of IPy/heparin (10 μM/0.7 μM) upon titration of protamine in HEPES buffer solution (10 mM, pH 7.4); (b) time-resolved decays of IPy/heparin (10 μM/0.7 μM) upon titration of protamine in HEPES buffer solution (10 mM, pH 7.4), inset: fluorescence decay lifetime data of IPy/heparin upon titration of protamine.
IPy/heparin upon titration of protamine. Obviously, it shows reversed ratiometric responses compared to IPy sensing heparin. The monomer intensity is significantly enhanced, at the same time the excimer emission is gradually reduced. An isoemissive point is observed at 471 nm. Clearly, this binary system also provides a ratiometric response to protamine. The green solution of the binary system changes back to blue upon addition of protamine (inset of Fig. 4a). The intensity ratio of monomer to excimer, IM/IE, is firstly gradually increased from 0.28 to 3.55 upon addition of protamine from 0 to 1.0 μM, and then abruptly and significantly increased to 165.10 upon further addition of protamine to 2.5 μM, which is ca. 600-fold enhancement. The further addition of protamine above 2.5 μM leads to no further enhancement of IM/IE, indicating the binding of protamine with heparin reaches saturation. Moreover, the ratiometric signal, IM/IE, of this binary sensor exhibits a good quadratic relationship with protamine concentration below 0.7 μM (inset of Fig. 4b). The corresponding equation is expressed as IM/IE = 0.280 (± 0.009) + 0.460 (± 0.071) [protamine] + 0.622 (± 0.134) [protamine]2 with the relative correlation coefficient of 0.997. Similar ratiometric response behaviors were also found for IPy/ heparin systems with different concentration of heparin (Fig. S7, SI). The DLs of these binary sensor systems were determined according to the standard 3σ criteria using their ratiometric responsive data (Fig. S8, SI), which are 15.4 nM (107.8 ng/mL), 25.8 nM (180.6 ng/mL), 27.2 nM (190.4 ng/mL), 77.6 nM (543.2 ng/mL), and 83.7 nM (585.9 ng/mL) for the binary systems containing heparin at 0.4, 0.5, 0.7, 0.8, and 1.0 μM, respectively. Clearly, low heparin concentration endows the binary system with high sensitivity, but leads to narrow responsive range. Although the sensitivity is not quite high compared to some reported sensors for protamine [8,19,26], the present sensor is, to the best of our knowledge, the only ratiometric sensor for protamine. Moreover, the response to protamine is fast and b1 min. 3.5. Selectivity of IPy/heparin to protamine To examine the selectivity of the binary system to protamine, the fluorescence response of IPy/heparin to different proteins and amino acids was measured. The examined proteins include poly-lysine, HIV-1 TAT, lysozyme (LYZ), trypsin (TPS), β-lactoglobulin (BLG), BSA, ovalbumin egg (O-egg), pepsin, and cytochrome c (Cyt-c). The measured positively charged amino acids include arginine (Arg), lysine (Lys) and histidine (His). As shown in Fig. 5a, among all the tested proteins at 2.5 μM, only protamine could produce prominent increases of the intensity ratio of IM/IE. Poly-lysine could also induce some ratiometric responses at this concentration, which is much smaller than that by protamine. TAT containing several Arg residues, and small positively charged amino acids (Arg, Lys, and His) could not produce apparent ratiometric responses. These results suggest that the large IPy-heparin
binding constant endow it with high selectivity to protamine that has even higher heparin-binding affinity. The high selectivity of the binary sensor system to protamine is also verified by the visual color change. As shown in the inset of Fig. 5a, only protamine and poly-lysine could change the green solution back to blue when exposed to 365 nm UV light, where the former leads to bright blue and the latter induces pale blue. The selectivity to protamine was further measured in the presence of these possible interferents. To this end, the ratiometric responses of the binary sensor were measured in the presence of one equiv. of each interferent. As shown in Fig. 5b, all the tested proteins or amino acids (except TPS and pepsin) display faint influence on the ratiometric responses to protamine. The presence of TPS and pepsin decreases the response extent to protamine. Even though, N10 times higher response to protamine is still observed than that without protamine (inset of Fig. 5b), indicating protamine could be detected even in the presence of TPS or pepsin. These results verify the feasibility of IPy/heparin binary sensor functioning as a ratiomertic sensor to protamine. 3.6. Sensing mechanism of IPy/heparin detecting protamine The process of IPy/heparin sensing protamine was also examined by UV–vis absorption and time-resolved decay measurements. As shown in Fig. 6a, along increasing protamine concentration, the blunt absorption bands of the binary system at 280, 350 and 380 nm gradually increase and become sharp accompanied with an obvious blue-shift, suggesting break-up of π-π stacking between pyrene moieties and giving rise to existence as monomer again. This reveals, to a certain extent, that heparin prefers to binding protamine to the probe due to the stronger affinity between protamine and heparin. As a result, the former electrostatic balance between the cationic probe and anionic heparin was broken, which leads to release of the probe from the anionic macromolecules (Scheme 3). As a control, the addition of lysozyme does not induce similar changes of UV–vis absorption of IPy/heparin (Fig. S9, SI), suggesting the electrostatic interaction between heparin and protamine plays an important role in the selective responses to protamine.
Scheme 3. Schematic representation of the process of IPy/heparin sensing protamine.
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Fig. 7. (a) Fluorescence emission of IPy (10 μM) in the absence and presence of heparin (0.3 and 2.0 μM); (b) color change of IPy solutions (10 μM) containing 0.3 and 2.0 μM heparin upon addition of different amount of protamine.
The time-resolved fluorescence decays of IPy/heparin upon titration of protamine are displayed in Fig. 6b, which were also monitored at 385 nm. The corresponding decay lifetimes are listed in the inset of Fig. 6b. It can be seen, along increasing protamine concentration, the two lifetimes of pyrene moieties change back to only one lifetime, suggesting the binding-state pyrene moieties change to non-binding state. Such results verify that the addition of protamine induces release of cationic probe from binding with heparin and gives rise to monomer emission, which undoubtedly was due to the stronger binding affinity of protamine with heparin.
free the probe from the complex with heparin and realize the blue color of the solution. To check the possibility of this methodology, we added protamine into the above two solutions and monitored the color change. As shown in Fig. 7b, the two solutions containing 0.3 μM and 2.0 μM heparin separately exhibit similar green color. The former needs 1.6 μM protamine to realize the solution color change back to blue, however, the latter needs ca. 10 μM protamine to accomplish the color change. Therefore, a proper differentiation between low concentrated heparin and high concentrated one could be realized by the subsequent added protamine.
3.7. Differentiation of heparin at low and high concentration
3.8. Ratiometric detection of heparin and protamine in serum solutions
As revealed earlier in Fig. 1a, the addition of heparin larger than 0.7 μM reduces the ratiometric responses as a result of dilution of aggregated pyrene probes with free heparins. Thus, a determined value of IE/ IM could be related with two different heparin concentrations: one is smaller than 0.7 μM and the other is larger than 0.7 μM. For example, as shown in Fig. 7a, the fluorescence emission of the probe (10 μM) changes to similar profile upon addition of 0.3 μM and 2.0 μM heparin. Moreover, the observed ratio of excimer to monomer is also similar (inset of Fig. 7a). Therefore, it is necessary to differentiate heparin at high concentration from that at low concentration. Considering the color change of IPy/heparin induced by protamine, it is possible to recognize the real heparin concentration by the amount of protamine used to reverse the solution color back to blue. This is because the binary system with high concentration of heparin should need more protamine to
Considering the important values of the potential application in detecting biological sample, we then measured the fluorescence responses of IPy to heparin and that of IPy/heparin system to protamine in bovine serum solution. As shown in Fig. 8a, the ratiometric response to heparin is also observed for IPy in aqueous buffer solution containing 0.2% bovine serum. Approximately 80-fold enhancement of IE/IM was obtained from 0.019 to 1.546 by addition of 1.0 μM heparin (Fig. 8b). We also measured the fluorescence responses of IPy to heparin in more concentrated serum solutions, such as 5% and 10% bovine serum. Similar ratiometric responses were observed again, where the monomer emission was decreased accompanied by the increased excimer emission upon continuous addition of heparin from 0 to 2 μM (Fig. S10, SI). It is just that the extent of the ratiometric responses is reduced in more concentrated serum solutions. Even though, a linear relationship still exists
Fig. 8. (a) Fluorescence spectra of IPy (10 μM) upon titration of heparin from 0 to 1.0 μM in 0.2% bovine serum solution; inset: fluorescence spectra of IPy (10 μM) upon titration of heparin from 1.0 to 10 μM in 0.2% bovine serum solution; (b) fluorescence intensity ratio of excimer to monomer (IE/IM) of IPy (10 μM) as a function of heparin concentration measured in 0.2% bovine serum solution.
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between the ratiometric response (IE/IM) and heparin concentration. We then checked the detection of protamine by the binary system based on IPy/heparin in biological solutions containing 0.2% and 2% bovine serum. Again, as in aqueous solution, a similar reversed ratiometric response expressed as decreased excimer and enhanced monomer was observed in serum solutions (Fig. S11 and S12, SI). Moreover, the ratiometric responses (IM/IE) of the binary system show linear relationship with protamine concentration below 0.8 μM in 2% serum solutions (inset of Fig. S12b). Such results suggest that applying the detection process of both IPy sensing heparin and IPy/heparin sensing protamine in biological samples is quite promising. 4. Conclusion The cationic imidazolium-modified pyrene derivative, IPy, can be used to function as a ratiometric fluorescent sensor for detection of heparin based on variation of monomer and excimer emission. The probe selectively exhibits remarkable monomer reduction along with excimer enhancement to the presence of heparin in HEPES buffer solution. The cationic probe has large heparin-binding affinity with binding constant of 3.9 × 106 M−1. In addition, the binary combination of IPy/heparin can realize highly sensitive and selective ratiometric detection of protamine among the tested proteins and positively charged amino acids. The larger heparin-binding affinity enables protamine to replace the heparinbound IPy and release the probe back to non-binding state, leading to enhanced monomer emission along decreased excimer emission. The detection limits of heparin and protamine were measured to be as low as 8.5 (153 ng/mL) and 15.4 nM (107.8 ng/mL), respectively. Moreover, the proposed method could be used for ratiometric detection of both heparin and protamine in biological samples. Acknowledgments The authors acknowledge the financial support from National Natural Science Foundation of China (21573140), Ministry of Education of China (NCET-12-0895), the Program of Introducing Talents of Discipline to Universities (B14041), the Program for Changjiang Scholars and Innovative Research Team in University (IRT_14R33). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.saa.2016.07.026.
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