Ratiometric Fluorescence Sensor Based on a Pyrene Derivative and

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Ratiometric Fluorescence Sensor Based on a Pyrene Derivative and Quantification Detection of Heparin in Aqueous Solution and Serum Qing Dai, Weimin Liu, Xiaoqing Zhuang, Jiasheng Wu, Hongyan Zhang, and Pengfei Wang* Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190, People’s Republic of China ABSTRACT: A ratiometric fluorescence sensor based on pyrene was designed for selective detection of heparin in HEPES (N-(2-hydroxyethyl)piperazine-N0 -ethanesulfonic acid) buffer and serum sample. Pyrene and long-chain alkanes were linked through bisquaternary functionality in the sensor which could interact with heparin via supramolecular assembly. A ratiometric fluorescent signal change of the sensor can be observed because of the specific monomer
excimer conversion of pyrene which is modulated by the supramolecular self-assembly of sensor and heparin. Upon addition of heparin, the excimer emission of the sensor at 489 nm is observed and the monomer emission intensity at 395 nm decreases concomitantly. Addition of heparin derivatives with very similar structure such as chondroitin 4-sulfate or hyaluronic acid to the same sensor solution only leads to very smaller changes in intensity ratios probably because of lower charge density and more distant spatial distribution of anions (or disadvantageous spatial orientation of anions) as compared to those of heparin. The novel sensor can effectively differentiate heparin from its derivatives with relatively low background interference and wide linear response in HEPES and serum. A linear calibration curve is obtained from 0 to 3.4 μM for heparin quantification in serum.

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eparin plays an important role in the regulation of various normal physiological and pathological processes such as cell growth and differentiation, inflammation, immune defense, lipid transport and metabolism, and blood coagulation.1
4 Clinically, heparin has been commonly used as an anticoagulant during cardiopulmonary surgery and to treat emergency deep venous thrombosis (DVT)5,6 based on its ability to accelerate the inactivation rate of coagulation factors such as thrombin and factor Xa.7 However, heparin overdose can induce some complications such as hemorrhages and heparin-induced thrombocytopenia.8,9 The therapeutic dosing level 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 longterm care.10,11 Therefore, close monitoring and quantification of heparin in serum is of vital importance for regulation of the physiological process and for clinical applications during surgery and the postoperative therapy period. To date, many methods have been established to monitor heparin concentration, including the activated clotting time, activated partial thromboplastin time, chromogenic antifactor Xa assay, and electrochemical and piezoelectric assays.12
15 However, these techniques are indirect and not sufficiently reliable, accurate, or amenable to clinical settings.16 Thus, the development of new methods with high accuracy and reliability is very much desirable. Recently, a variety of fluorescent17
26 and colorimetric11,23,27
29 methods that are highly sensitive and simple have been reported for heparin sensors. Some cationic chromophores, such as tripodal boronic acids,17 polycationic calix[8]arenes,18 and a chromophore-tethered flexible copolymer,19 r 2011 American Chemical Society

have been reported as heparin sensors. Most of these assays adopt fluorescence quenching as the signal output, which is often unfavorable for heparin detection because of large environment effects or low sensitivity. Subsequently, peptide-based sensor,20 cationic silole derivative,22 and cationic conjugated polyelectrolytes29 were reported to show enhanced fluorescence upon interaction with heparin. However, one common feature of these reported fluorescent sensors of heparin is that they use the single emission intensity change as the detection signal. Such sensors tend to be affected by a variety of factors such as instrumental efficiency, environment conditions, and sensor molecule concentration. Ratiometric sensors can effectively eliminate most or all interferences from environment by built-in correction of two emission bands, so they have been widely used in complex biological samples.30
32 Pyrene is often used as the fluorophore of ratiometric sensors, which can form an excimer when an excited-state molecule is brought into close proximity with another groundstate molecule.33,34 On the basis of these unique photophysical properties of pyrene and the highly sulfated polysaccharide structure of heparin, we designed and synthesized a dual quaternary ammonium 1,4-diazobicyclo(2,2,2)octane (DABCO) derivative 1 (Scheme 1). Electrostatic and hydrophobic effects were used to control the supramolecular self-assembly process. The sensor molecule 1 showed a ratiometric fluorescence response for heparin in HEPES buffer solution and serum with Received: April 6, 2011 Accepted: July 29, 2011 Published: July 29, 2011 6559

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Analytical Chemistry Scheme 1. Chemical Structure and Synthetic Route of Compound 1

remarkably high selectivity and sensitivity. Moreover, 1 exhibited a wide quantification range for heparin (0
3.4 μM) in a diluted serum sample (5% serum) which may find applications in related research fields requiring heparin quantification.

’ EXPERIMENTAL SECTION Reagents and Apparatus. Pyrene-1-carbaldehyde was purchased from Alfa Aesar Co. Heparin and bovine serum albumin (BSA) were provided by Biodee, China. All analytical chemicals were purchased from Sigma Company for direct use, including chondroitin sulfate (Chs) and hyaluronic acid (HA). Other chemicals are of analytical reagent grade, purchased from Beijing Chemical Regent Co. All of them were used directly without further purification. The water used throughout all experiments was purified by a Millipore filtration system. Methanol was dried with magnesium chips and then distilled under reduced pressure. The molecular weight of heparin was determined by disaccharide (644.2 g/mol), and its concentration was calculated using disaccharide as the repeat unit. All solvents used for spectra measurement are of chromatographic grade. The stock solutions of heparin (1.0 mM) in 10.0 mM HEPES buffer solution (pH = 7.4) and sensor 1 (1.0 mM) in ethanol were prepared for spectral measurements. Melting point was tested on the X-4 microscopic melting point apparatus bought from Beijing Taike Co. 1H NMR and 13C NMR spectra were measured on a Bruker Avance Π-400 spectrometer using DMSO and/or CDCl3 as the solvent and tetramethylsilane (TMS) as an internal reference. Mass spectrometry (MS) and high-resolution mass spectrometry (HR-MS) spectra were recorded on a Waters GCT Premier mass spectrometer. UV
vis spectra and fluorescence spectra were obtained with Hitachi U-3010 and F-4500 spectrophotometers, respectively. The pH measurements were carried out on a MettlerToledo Delta 320 pH meter. Heparin Detection in Buffer Mixture Solution. The stock solution of sensors 1 in ethanol (1 mL, 1.0 mM), 0.5 mL of ethanol, and 8.5 mL of HEPES buffer water solution (10 mM, pH 7.4) were mixed together to yield testing used buffer solution (100 μM of 1, ethanol/water (15/85, v/v)). Then heparin stock solution (1.0 mM) was added dropwise into 2 mL of the above mixture solution for spectral analysis. Upon each addition, the

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mixture was gently shaken before UV
vis and fluorescence analysis. The absorption spectra and fluorescence spectra were collected in the range of 250
450 and 350
650 nm, respectively, with an excitation wavelength of 345 nm. Heparin Detection in Fetal Bovine Serum (FBS). The stock solution of sensors 1 in ethanol (0.5 mL, 1.0 mM), 1.0 mL of ethanol, 0.5 mL of FBS, and 8.0 mL of HEPES buffer solution (10 mM, pH 7.4) were mixed together to yield the final testing used solution (50 μM of 1, ethanol/water (15/85, v/v) containing 5% FBS). Then, heparin stock solution (1 mM) was added dropwise into 2 mL of the above mixture solution for spectral analysis. Synthesis of Compound 2. 1-Bromotetradecane (2.92 mL, 9.79 mmol) was added into the solution of 1,4-diazobicyclo(2,2,2)octane (DABCO) (2.0 g, 17.8 mmol) in 30 mL of methanol. Then the mixture was stirred at 60 °C for 6 h. After cooling, the solvent was removed under reduced pressure. Then CH2Cl2 was added, and the mixture was purified by column chromatography (CH2Cl2/CH3OH = 5:1). Evaporation of the solvent afforded 2 (6.23 g, 90%) as a pale white solid. Mp: 152 °C. 1H NMR (DMSO, 400 MHz,) δ (ppm): 0.85 (t, 3H), 1.24 (m, 22H), 1.64 (m, 2H), 3.01 (t, 6H), 3.18 (m, 2H), 3.27 (t, 6H). 13C NMR (CDCl3, 100 MHz) δ (ppm): 13.9, 21.1, 22.1, 25.9, 28.6, 28.8, 28.9, 29.1, 31.3, 44.7, 51.3, 63.0. ESI-MS m/z: M+, calcd for C20H41N2, 309.3; found, 309.3. Synthesis of Compound 4. 1-Pyrenemethanol (3) was synthesized according to a reported method.24 1,4-Dibromobutane (1.4 mL, 11.8 mmol) was added to a stirred mixture of 3 (0.90 g, 3.88 mmol) and NaOH (0.386 g, 9.65 mmol) in water (30 mL). Then CTAB (0.04 g, 0.097 mmol) was added, and the mixture reacted at 70 °C overnight. After cooling, CH2Cl2 (30 mL) was added and the mixture was separated, then the water phase was extracted with CH2Cl2 (20 mL  2). The combined organic phase was washed with saturated brine and dried with MgSO4. The concentrated mixture was purified by column chromatography (petroleum ether/dichloromethane = 1:1) to give 4 (1.21 g, 85%). Mp: ∼215 °C. 1H NMR (CDCl3, 400 MHz,) δ (ppm): 1.82 (m, 2H), 1.98 (m, 2H), 3.41 (t, 2H), 3.64 (t, 2H), 5.22 (s, 2H), 8.00
8.07 (m, 4H), 8.14
8.22 (m, 4H), 8.36 (d, 1H). Synthesis of Compound 1. 2 (0.39 g, 1.0 mmol) was added to a solution of 4 (0.36 g, 0.98 mmol) in methanol (20 mL). The mixture was heated to reflux under a nitrogen atmosphere for 48 h. After cooling, the precipitates were filtrated. The crude products were recrystallized with methanol and gave pale white powders (0.63 g, 85%). Mp: 220 °C. 1H NMR (DMSO, 400 MHz,) δ (ppm): 0.85 (t, 3H), 1.24 (m, 22H), 1.64
1.66 (m, 4H), 1.80 (s, 2H), 3.47 (t, 2H), 3.54 (t, 2H), 3.67 (t, 2H), 3.81 (s, 12H), 5.23 (s, 2H), 8.08
8.13 (m, 2H), 8.20 (s, 2H), 8.27
8.35 (m, 4H), 8.40 (d, 1H). 13C NMR (DMSO/CDCl3, 100 MHz) δ (ppm): 13.9, 18.8, 21.4, 22.1, 25.6, 26.0, 28.5, 28.7, 28.8, 29.0, 29.1, 31.3, 50.3, 54.5, 63.3, 63.5, 68.9, 70.6, 123.4, 123.9, 124.1, 124.4, 125.2, 125.2, 126.1, 127.0, 127.2, 127.5, 128.7, 130.2, 130.6, 130.7, 131.6. HR-MS m/z: (M
1)+, calcd for C41H60N2O, 595.4617; found, 595.4606.

’ RESULTS AND DISCUSSION Structure of Sensor 1 and the Polysaccharide Targets. The synthetic route for fluorescent sensor 1 is depicted in Scheme 1. The chemical structures of polysaccharides are shown in Figure 1. Sensor 1 is a dual quaternary ammonium salt molecule with a 6560

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Analytical Chemistry pyrene chromophore and a long alkyl chain attached at the two nitrogen atoms of the DABCO unit. The chondroitin 4-sulfate (Chs) and hyaluronic acid (HA) are often the main contaminates of clinical heparin, so they were selected to evaluate the performance of sensor 1. Heparin (Hep) is a highly sulfated glycosaminoglycan and is composed of a 1f4-linked iduronic acid and glucosamine repeating disaccharide unit. On average, heparin carries three sulfate groups (2-O-sulfation of iduronic-acid, 2-Nsulfation, and 6-O-sulfation of glucosamine) and one carboxylate group per disaccharide unit, among which one carboxylate and sulfate group locate at the same side of one saccharide ring. Chs consists of the disaccharide unit formed by 1f3-linked Nacetylgalactosamine and glucuronic acid, modified by sulfation

Figure 1. Chemical structures of Hep, ChS, and HA.

Scheme 2. Schematic Illustration of Heparin Detection Using Sensor 1

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in position 4. Chs possesses one sulfate and one carboxylate moiety per disaccharide unit. HA is formed by a 1f3-linked glucuronic acid and N-acetylglucosamine, which has only one carboxyl group per disaccharide unit. Therefore, the dual positively charged DABCO unit can effectively form electrostatic interactions with the negatively charged heparin and bring two molecules of sensor 1 into one saccharide ring. The long hydrophobic chain may induce the directional alignment of sensor 1, which results in the formation of a dimer structure to give an excimer emission. The schematic illustration of heparin detection is shown in Scheme 2. Chs and HA should not induce an efficient excimer emission because of lower charge density and/or disadvantageous spatial orientation of anions. Heparin Detection in HEPES Buffer Solution. We first investigated the interaction of sensor 1 with heparin in HEPES buffer solution (water/ethanol, 85/15, v/v). In a solution of water/ethanol (85/15), 1 (100 μM) displays an absorption band with several peaks at 275, 326, and 342 nm corresponding to the pyrenyl monomer band (Figure 2a). Upon binding with heparin, a significant red shift is observed as shown in Figure 2a. The band broadening and red shift in the UV spectra of 1 upon the addition of heparin are attributed to the favorable intermolecular π
π stacking of the two pyrene moieties in the ground state.35 This provides evidence for the formation of an intermolecular excimer of 1 when it interacted with heparin. The excimer formation can be observed more directly from the fluorescence spectra (Figure 2b). In absence of heparin, 1 shows a characteristic monomer emission with peaks at 375 and 395 nm, and no observable excimer emission could be found. Upon addition of heparin, a broad and structureless emission band centered at 489 nm is observed, and the fluorescence emission intensity at 395 nm decreases concomitantly. The emission band at 489 nm is attributed to the formation of an excimer by pyrene and shows significant enhancement with increasing concentration of heparin. From Figure 2b, it can be found that sensor 1 can rationally identify heparin in buffer solution (10 mM, HEPES) through a ratiometric fluorescence approach. The emission intensity ratio, I489/I395, gradually increases from 0.015 to 1.3 as the concentration of heparin varies from 0 to 30 μM (Figure 2c). The inset of Figure 2b shows the photoimage of sensor 1 (100 μM) in water/ ethanol (85/15, v/v) buffer solution when heparin is absent (A) or present (30 μM) (B). The strong “turn-on” excimer emission can be seen by naked eyes under a UV lamp.

Figure 2. Absorption (a) and fluorescence (b) spectra of 1 (100 μM) in the presence of different concentrations of heparin (0, 3, 7, 10, 14, 18, 22, 26, 30 μM) in buffered (10 mM, HEPES, pH 7.4) water/ethanol (85/15, v/v) solution (λex = 345 nm). The inset shows the photographs of 1 (100 μM) solution at 0 (A) and 30 μM (B) heparin in buffer at room temperature. (c) The changes of the fluorescence intensity ratio of sensor 1 (I489/I395) vs the heparin concentration. 6561

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Figure 3. Effect of pH on the fluorescence intensity ratio (I489/I395) of free 1 (100 μM) (black squares) and 1/heparin (30 μM) mixtures (red dots) at room temperature.

Figure 4. Selectivity of 1 (100 μM) to heparin (30 μM) over competing anions or biological molecules in a water/ethanol (85/15, v/v) buffer solution. Interfering species: (0) none, (1) Na2SO4, (2) Na3PO4, (3) CH3COONa, (4) sodium citrate, (5) ATP, (6) glucose, (7) BSA, (8) HA, (9) Chs, (10) heparin. BSA was 0.5 mg/mL, and the other anions or biological molecules were 30 μM.

Effect of pH on the Performance of Sensor 1. To further

study the above mechanism and the practical applicability of the method, the effects of pH on the fluorescent response to heparin of the new sensor 1 were also investigated. Experimental results show that the intensity ratios of free 1 have no significant changes in a wide pH range from 4 to 11 (Figure 3). However, upon addition of heparin, 1 shows different intensity ratio enhancements. Under acidic conditions (pH < 6), the carboxylate and sulfate groups of heparin may be partially protonated, which decreases the electrostatic interactions of sensor and heparin. At pH over 9, quaternary ammonium cations (Nc+) of 1 may form an NcOH complex, which can weaken the electrostatic interactions of sensor 1 and heparin.36 Sensor 1 shows the highest fluorescence response toward heparin in the pH range of 6.0
8.0. Therefore, the pH value of 7.4, near physiological conditions, was chosen as the optimum experimental condition. Selectivity. Selectivity is a very important parameter to evaluate the performance of a new fluorescence sensor. Particularly, for a sensor with potential applications in biomedical sampling, a highly selective response to the target over other potentially competing species is a necessity. Therefore, the selectivity experiments for sensor 1 were extended to various anions including some biological important molecules, sodium

Figure 5. (a) Fluorescence spectra of 1 (100 μM) in 5% FBS containing buffered (10 mM, HEPES, pH 7.4) water/ethanol (85/15) solution upon addition of 0
22.5 μM heparin at an interval of 2.5 μM. (b) Intensity ratio (I489/I395) as a function of heparin concentration. The inset shows the photographs of a solution of 1 after addition of 0 (A) and 22.5 μM (B) heparin in 5% serum containing buffer at room temperature.

sulfate, sodium phosphate, sodium acetate, adenosine triphosphate, glucose, sodium citrate, and BSA. In addition, Chs and HA, analogues of heparin, were also investigated. The fluorescence intensity ratios (I489/I395) were tested to evaluate the selectivity of sensor 1. As shown in Figure 4, none of these anions or biological molecules cause interferences although citrate, BSA, Chs, and HA induce a slight increase in the intensity ratio. As shown in Figure 4, the selectivity of 1 is in the order heparin . Chs > HA, suggesting that electrostatic interactions (high charge density of heparin) and the conformation of the sugar dimer play a dominant role in binding, which is also consistent with the sensor mechanism proposed above. All these results indicate that our proposed sensor 1 could meet the selective requirements for heparin detection. Heparin Detection in Fetal Bovine Serum Medium. Sensor 1 showed good sensitivity and selectivity for heparin in pure buffer, but to be more useful in a bioassay, the detection system should be able to tolerate any interference from real biological samples. Therefore, FBS was used to investigate the feasibility of the method for use with biological samples. Figure 5 shows the spectra of the sensor 1 in 5% FBS containing buffered water/ ethanol (85/15) solution. As shown in Figure 5a, addition of heparin to a 50 μM solution of 1 results in a gradual increase of excimer fluorescent intensity and decrease of monomer 6562

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Figure 6. ln(S/B) of sensor 1 as a function of the heparin concentration in pure buffer solution (a) and in 5% FBS containing solution (b).

fluorescent intensity. The saturation of the intensity at 489 nm occurs at [heparin] = 22.5 μM. Further addition of heparin will lead to decrease of monomer and excimer fluorescent signals (not shown). This can be attributed to the fact that each disaccharide unit of excess heparin can only interact with less than two or one molecule of 1 and block the excimer formation. The heparin saturation concentration in serum is lower as compared to that shown in Figure 2b. This is due to charge pairing between the proteins in serum and 1, which partially blocks the interaction between 1 and heparin. Figure 5b shows the fluorescence intensity ratio as a function of heparin concentration. The original ratio of free 1 in serum (0.11) is higher than that of 1 in buffer (0.015), which indicates higher background fluorescence of 1 in serum. However, in a solution containing a low concentration of serum, it would not cause interference for heparin detection. As shown in the inset of Figure 5, distinguishable fluorescent colors are observed for 1. These results clearly indicate that the cationic sensor 1 could achieve the naked detection of heparin in serum through use of an ultraviolet lamp. Heparin Quantification. As discussed above, sensor 1 can effectively identify heparin in both a HEPES solution and serum sample through a ratiometric approach. To further eliminate the background signal and quantify heparin, we defined signal-tobackground ratio (S/B) using the following equation:37,38 ðI489 =I395 Þwith heparin S=B ¼ ð1Þ ðI489 =I395 Þno heparin where (I489/I395)with heparin and (I489/I395)no heparin take into account the ratio of excimer fluorescence intensity to monomer fluorescence

Figure 7. (a) Fluorescence spectra of 1 (20 μM) in 5% FBS containing buffered (10 mM, HEPES, pH 7.4) water/ethanol (90/10) solution upon addition of 0
6.0 μM heparin at an interval of 0.2 μM. (b) Intensity ratio (I489/I395) as a function of heparin concentration. (c) S/B of sensor 1 (20 μM) as a function of the heparin concentration in 5% FBS containing solution.

intensity of pyrene with and without heparin, respectively. So we can find that the signal-to-background ratios of 1 are relatively high, which are 86.7 at 30 μM heparin in buffer and 14.4 at 22.5 μM heparin in serum sample. Figure 6 shows ln(S/B) as a function of the heparin concentration. There is a good linearity between the ln(S/B) and concentration of heparin in the range of 5
30 μM in HEPES and from 0 to 22.5 μM in serum (the correlation coefficients are 0.998 and 0.999, respectively). The detection limits in buffer and serum, defined as 3 times the standard deviation of background, are 0.157 and 0.53 μM, respectively. Taking into account of that the testing serum sample is 5% serum containing, the linear 6563

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Analytical Chemistry response range of 0.53
22.5 μM (0.063
2.6 U/mL) for heparin in the serum samples should be corresponding to 10.6
450 μM (1.26
52 U/mL) in pure serum sample, which can satisfy the requirements of clinical correlative heparin monitoring during cardiovascular surgery (17
67 μM). Moreover, by changing the sensor concentration and water fraction, a lower detection limit could be obtained. To extend the heparin calibration concentration to the whole clinical correlative range, a lower concentration of 1 (20 μM) and a higher water fraction (90%) were utilized for heparin quantification.23,37 Figure 7a shows the spectra of the sensor 1 (20 μM) in 5% FBS containing buffered water/ethanol (90/10) solution. The saturation of the intensity at 489 nm occurs at [heparin] = 6.0 μM. As shown in Figure 7b, the original ratio of free 1 (20 μM) in serum (0.0717) is lower than that of 1 (100 μM) (0.11), which should be partially attributed to the decreased fluorescence background at dilute sensor concentrations. A good linearity between the S/B and concentration of heparin was achieved from 0 to 3.4 μM in serum (the correlation coefficients are 0.999) (Figure 7c), and the detection limit was 0.068 μM. The linear response range in pure serum sample was corresponding to 1.3
68 μM (0.16
8.2 U/mL), which can cover the whole clinical correlative range. These results suggest that sensor 1 is potentially appropriate for highly sensitive quantification of heparin content in serum samples.

’ CONCLUSION In summary, we have successfully developed a fluorescence ratiometric sensor for quantification detection of heparin by taking advantage of the unique change of fluorescence intensity ratio based on supramolecular assembly with distance sensitivity. Electrostatic interaction and hydrophobic effects between the sensor and heparin are used to regulate the supramolecular assembly process. The fluorescence sensor shows excellent selectivity for heparin with a large contrast of fluorescence in buffer solution. Moreover, a good linear response for heparin was found in the range of 0
3.4 μM (approximately 0.41 U/mL) in 5% serum sample. The assay reported herein may find potential applications in research requiring rapid detection and quantification of purified heparin samples or in biological media. ’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Fax: +86-10-62554670. Phone: +86-10-82543512.

’ ACKNOWLEDGMENT This work was supported by the Main Direction Program of Knowledge Innovation of Chinese Academy of Sciences, the NNSF of China (Nos. 21073213, 20903110), the Research Grants Council of the Hong Kong SAR (No. CityU 123607), and the “863” Program (No. 2009AA03Z318).

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