Biosensors and Bioelectronics 55 (2014) 174–179
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Ultrasensitive fluorescence detection of heparin based on quantum dots and a functional ruthenium polypyridyl complex Yanlin Cao, Shuo Shi n, Linlin Wang, Junliang Yao, Tianming Yao n Department of Chemistry, Tongji University, 1239 Siping Road, Shanghai 200092, China
art ic l e i nf o
a b s t r a c t
Article history: Received 6 September 2013 Received in revised form 21 November 2013 Accepted 2 December 2013 Available online 12 December 2013
A new strategy for the detection of heparin is developed by utilizing quantum dots (QDs) and a functional ruthenium polypyridyl complex [Ru(phen)2(dppz-idzo)]2 þ (phen ¼ 1,10-phenanthroline, dppz-idzo ¼dipyrido-[3,2-a:20 ,30 -c] phenazine-imidazolone). The emission of CdTe QDs is found to be quenched by Ru complex due to electron transfer. Upon addition of the polyanionic heparin, it could remove the quencher (Ru complex) from the surface of QDs owing to the electrostatic and/or hydrogen bonding interactions between heparin and Ru complex, which led to significant fluorescence recovery of CdTe QDs. The fluorescence intensity enhanced with the increase of heparin and a linear relationship was observed in the range of 21–77 nM for heparin detection in buffer solution and the limit of detection (LOD) is 0.38 nM. Moreover, the strategy was successfully applied to detect heparin as low as 0.68 nM with a linear range of 35–98 nM in fetal bovine serum samples. The selectivity results of the fluorescence assay revealed that our system displayed excellent fluorescence selectivity towards heparin over its analogues, such as chondroitin 4-sulfate (Chs) or hyaluronic acid (Hya). This fluorescence “switch on” assay for heparin is label-free, convenient, sensitive and selective, which can be used to detect heparin in biological systems even with the naked eyes. & 2013 Elsevier B.V. All rights reserved.
Keywords: Quantum dots Ru complex Fluorescence Heparin Fetal bovine serum
1. Introduction Heparin is a highly negatively charged sulfated linear polymeric glycosaminoglycan (Capila and Linhardt, 2002a, 2002b), it has been clinically used in different forms in anticoagulation treatment during cardiopulmonary surgery and in emergency deep venous thrombosis conditions (Shriver et al., 2000). While, the overdose of heparin often causes potentially fatal bleeding complication such as hemorrhages and thrombocytopenia (Girolami and Girolami, 2006). So it is of crucial importance to monitor closely heparin levels for the sake of health. So far many efforts have been devoted to the detection and quantification of heparin. The conventional methods for heparin detection include activated partial thromboplastin time (Cheng et al., 2001), activated clotting time (Bowers and Ferguson, 1994), potentiometric assays (Langmaier et al., 2007) and ion pair high performance liquid chromatography (Patel et al., 2009). However, these methods exist some problems such as the lack of specificity and potential interference from other factors. Recently, more researches have focused on the direct detection of heparin by binding to sensing device or probe, such as PEI/AuNPs as a probe for the colorimetric detection of heparin (Wen et al., 2013), GO quenching the color of self-assembly
n
Corresponding authors. Tel./fax: þ86 21 65983292. E-mail addresses: [email protected] (S. Shi), [email protected] (T. Yao).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.12.009
gold nanorods (Fu et al., 2012b) and gold nanoparticles (Fu et al., 2012a), target-induced self-assembly of polyethyleneimine capped Mn-doped ZnS quantum dots (Yan and Wang, 2011), adenosinebased molecular beacons as light-up probes (Kuo and Tseng, 2013), mallard blue as a sensor via simple UV–vis measurements (Bromfield et al., 2013), norfloxacin–cerium complex as a spectofluorimetric sensor (Patil et al., 2009) and so on. Among these methods, fluorescent chemosensors received intense attention recently, however, some of them have limitations including poor sensitivity and selectivity. Heretofore, ultrasensitive fluorescence “switch-on” sensors for heparin, which have good selectivity, and can be used for direct monitoring of heparin in serum, still remain rare. Therefore, there is still a high demand for a convenient, sensitive and rapid method for recognizing and sensing of heparin. In addition, ruthenium polypyridyl complexes have been widely investigated because of their prominent DNA-binding properties (Friedman et al., 1990; Jenkins et al., 1992). Our laboratory has also studied the binding behaviors of many ruthenium complexes with various DNA structures (Shi et al., 2010a, 2010b, 2012a, 2012b, 2013a, 2013b; Sun et al., 2011). However, few has been reported heretofore about the interaction between Ru(II) complexes and heparin. Rozenberg first used a ruthenium complex [Ru(2,20 -bipyridine)3]2 þ for the detection of heparin in gels by fluorescent staining (after electrophoresis, isolation, incubation and washing) (Rozenberg et al., 2001). But this complex does not respond to heparin in buffered solution or serum containing samples. Later, Szelke et al. (2009) attached
Y. Cao et al. / Biosensors and Bioelectronics 55 (2014) 174–179
cationic residues to the bipyridine ligands and got a polycationic fluorescent ruthenium complex. They succeeded to detect heparin in buffer solution or serum. However, this “switch-off” detection suffered from the low sensitivity and specificity. In our recent study, a new strategy for heparin detection and quantification in biological media, such as fetal bovine serum (FBS), was developed by monitoring the emission change of a ruthenium polypyridyl complex [Ru (phen)2(dppz-idzo)]2 þ in buffer solutions (Cheng et al., 2013). [Ru (phen)2dppz-idzo]2 þ was almost non-emissive in aqueous solution due to the hydrogen bonding between the solvent and the phz (phenazine) nitrogens on the main ligand (dppz-idzo) (Yao et al., 2013). However, in the presence of heparin, the complex was supposed to interact with the sulfanilamido, sulfate and carboxylate groups on the pyranosyluronic acid unit in heparin through electrostatic and/or hydrogen bonding interactions and got protected from water, resulting in excimer emission of complex [Ru(phen)2dppzidzo]2 þ . Thus, our complex could be employed for the fluorescence “switch on” detection of heparin. However, the sensitivity of this method was unsatisfied, the limit of detection (0.011 U mL 1, or 10.5 nM calculated according to the heparin molecular mass of 6000 g mol 1) was high. In order to solve these problems, we designed another strategy to detect heparin based on CdTe quantum dots and Ru complex [Ru(phen)2(dppz-idzo)]2 þ . Quantum dots (QDs), one of the most attractive nanometer-sized luminescent semiconductor crystals, have many advantages over organic dyes such as excellent photostability, high quantum yield, tunable emission wavelength, and high photobleaching threshold (Algar et al., 2011). In addition, the large surface of QDs is favorable for attaching variable ligands and thus getting controllable properties (Smith et al., 2008). Up to now, QDs have been widely used as novel luminescence indicators of different biological processes and bioanalysis in recent years (Chen et al., 2013; Guo et al., 2013; Li et al., 2013). The interactions between QDs and Ru polypyridine complexes in solution have been reported (Sykora et al., 2006). The emission of QDs could be quenched by Ru complexes and the possible mechanism for QDs emission quenching is the photo induced electron transfer (Sun et al., 2013). In our laboratory, we have proposed label-free, aptamer-based biosensors for the selective detection of thrombin (Sun et al., 2012) and silver ions (Sun et al., 2013) by using a molecule “light switch” Ru polypyridine complex [Ru(bpy)2(dppz)]2 þ and QDs. Herein, we designed a label free, sensitive and selective luminescence sensor for heparin based on CdTe quantum dots and Ru complex [Ru(phen)2(dppz-idzo)]2 þ (Scheme 1). Upon addition of the Ru complex, the positively charged Ru complex and negatively charged water-soluble CdTe quantum dots form an anionic conjugate due to their electrostatic attraction in aqueous solution. This static association between the Ru complex and QDs induces ultrafast photoinduced electron transfer from QDs to the electron acceptor (the Ru complex), preventing the normal recombination of the electron and the hole in QDs, and results in a decrease of the fluorescent intensity of QDs. When added into the mixture, heparin could remove Ru complex (the quencher) from the surface of QDs due to its high avidity for Ru complex, and the fluorescence of QDs could be recovered. This sensing platform provides an ultrasensitive, simple, and selective method for the detection of heparin.
2. Materials and methods 2.1. Materials Heparin, NaBH4, tellurium powder, CdCl2, thioglycolic acid and NaOH was purchased from Sigma-Aldrich. Glucose, glycine, adenosine triphosphates (ATP), uridine monophosphate (UMP), bovine serum albumin (BSA), chondroitin sulfate (Chs), hyaluronic
175
acid (Hya), and fetal bovine serum (FBS) were obtained from Genestar, Shanghai, China. [Ru(phen)2dppz-idzo)]2 þ was synthesized by our laboratory according to the literature (Yao et al., 2012). Heparin sodium salt was got from porcine intestinal mucosa and 1 μM heparin corresponded to 6 μg/mL (Molecular mass: 6.0 kDa). Stock solutions of heparin, Chs, and Hya in water were prepared and stored at 4 1C before use. Fetal bovine serum was stored at 20 1C and was brought to room temperature when used. Tris–HCl buffer solution (10 mM Tris, pH 7.4) was used in all of the experiment. 2.2. Instrumentation The fluorescence spectra were recorded at room temperature on an F-7000 fluorescence spectrophotometer (Hitachi) with a quartz cell (1 mm). Both the excitation and emission slit widths were fixed at 10 nm. Lambda Bio 40 UV/Vis Spectrophotometer (Perkin-Elmer, USA) was used to quantify the QDs. 2.3. Preparation of CdTe QDs The synthesis of CdTe QDs was performed according to the reference with some modification (Peng et al., 2007) (In electronic supplementary information). The UV–vis spectrum of TGA (thioglycolic acid)-capped CdTe QDs was obtained (Fig. S1). The sizes and concentrations of QDs can be estimated from the adsorption peaks in UV–vis spectrum (Yu et al., 2003). 2.4. Optimization of the sensor For the detection of the quenching behavior of [Ru(phen)2(dppzidzo)]2 þ on the fluorescence of QDs, 10 μL CdTe QDs and various amounts of [Ru(phen)2(dppz-idzo)]2 þ were incubated in 10 mM Tris–HCl buffer (pH 7.4) at room temperature for 5 min in a 1.5 mL Eppendorf tube. The final concentration of CdTe QDs was 88 nM. Then the fluorescence spectra were measured in a 2 mL quartz cuvette at room temperature. In order to obtain a highly sensitive response for the detection of heparin, the optimization of the conditions including buffer solution and pH value were carried out in our experiment. We selected H2O, AC–NH4, HEPES, MOPS-NH2OH, PBS, Tris–AC, Tris– HCl as buffer solutions. A sample of 10 μL CdTe QDs and 70 μL [Ru(phen)2(dppz-idzo)]2 þ (20 μM) were incubated for 5 min in different buffer solutions. Next, 80 μL heparin (0.7 μM) was mixed with this solution and incubated at room temperature for another 5 min. The resulting solutions were studied by fluorescent spectroscopy at room temperature with excitation at 365 nm, both the excitation and emission slit widths were 10 nm. In the same way, the optimization of pH value was carried out in Tris–HCl buffer solution with different pH values. 2.5. Fluorescence detection of heparin in buffer solution For the fluorescence assay of heparin, 10 μL CdTe QDs and 70 μL [Ru(phen)2(dppz-idzo)]2 þ (20 μM) were incubated for 5 min in 10 mM Tris–HCl buffer (pH 7.4). Next, different concentration of heparin was mixed with this solution and incubated at room temperature for another 5 min. The final concentration of Ru complex was 1.4 μM, CdTe QDs was 88 nM. Then the fluorescence spectra were measured. 2.6. Analysis of heparin in fetal bovine serum All fluorescence assays were carried out in biological media, 1% fetal bovine serum (FBS) in 10 mM Tris–HCl buffer.
176
Y. Cao et al. / Biosensors and Bioelectronics 55 (2014) 174–179
Scheme 1. Schematic description of fluorescence detection of heparin by utilizing CdTe quantum dots and Ru complex [Ru(phen)2(dppz-idzo)]2 þ and chemical structures of Chs, Hya, heparin.
2.7. Sensor selectivity investigation In the selectivity experiment, 10 μL CdTe QDs and 70 μL [Ru (phen)2(dppz-idzo)]2 þ (20 μM) were incubated for 5 min in 10 mM Tris–HCl buffer (pH 7.4). Next, a series of biomolecules, including glucose, glycine, adenosine triphosphates (ATP), uridine monophosphate (UMP), bovine serum albumin (BSA), chondroitin sulfate (Chs), hyaluronic acid (Hya) were mixed with this solution and incubated at room temperature for another 5 min. The concentration of heparin was 100 nM, the other biomolecules was 1 μM, CdTe QDs was 88 nM and Ru complex was 1.4 μM. The resulting solutions were studied by fluorescent spectroscopy at room temperature with excitation at 365 nm, both the excitation and emission slit width were 10 nm.
3. Results and discussion 3.1. Optimization of the sensor To investigate the quenching behavior of [Ru(phen)2(dppzidzo)]2 þ on the fluorescence of CdTe QDs, the fluorescence signals toward different concentrations of Ru complex were measured in 10 mM Tris–HCl buffer (pH 7.4). The fluorescence intensity decreased with increasing of Ru complex and the emission intensity plotted against the concentration of Ru complex were shown in Fig. 1A and Fig. 1B, respectively. The free QDs show strong fluorescence emission in Tris–HCl buffer. When 1.4 μM Ru complex was added, the fluorescence intensity of QDs (88 nM) could decrease to 0.57%, indicating Ru complex could quench the fluorescence of QDs by electronic interactions that occurred upon attachment. We figured out that the binding constant of Ru complex for QDs was 5.53 105 (In electronic supplementary information). The high quenching efficiency would lead to a high signal-to-back ground ratio and good sensitivity for target detection. In our later experiments for heparin detection, 1.4 μM Ru complex was selected to ensure high quenching efficiency. QDs and Ru complex could interact and then formed a fluorescencequenched complex which was used for analytical purposes, named QDs–Ru, hereafter. Buffer solution and pH values can not only affect the fluorescence intensity of QDs, but also affect the interaction between [Ru (phen)2(dppz-idzo)]2þ and heparin. For this reason, it is important to select an appropriate buffer and pH value for assay of heparin. Here,
six kind buffer solutions including AC–NH4, HEPES, MOPS–NH2OH, PBS, Tris–AC, Tris–HCl were investigated (Fig. S3). The concentration of each buffer was 10 mM and the pH was 7.4. The result (Fig. 1C) shown that the fluorescence intensity in Tris–HCl was higher than that in other five buffers. Thus, Tris–HCl was selected in this study for all other experiments. As for the pH value, the effect of pH within a range from 3.5 to 8.5 was investigated (Fig. S4). As shown in Fig. 1D, the fluorescence intensity increased gradually with the pH ranging from 4.0 to 8.5, but the increase from 7.5 to 8.5 was not so obvious. The pH value in the body is 7.35–7.45 and pH 7.4 is a mild condition, therefore, pH 7.4 was selected in this study.
3.2. The analytical performance of the sensor The free [Ru(phen)2(dppz-idzo)]2 þ has no fluorescence, but it can emit fluorescence at 630 nm after binding to heparin (Fig. S2). However, the fluorescence intensity is much lower than that of QDs and the fluorescence emission has a red shift in our system (Fig. 1). Thus, we mainly focused on the fluorescence intensity of QDs in our experiments. To investigate our strategy for the detection of heparin, the emission of QDs–Ru as a function of heparin concentration was measured. The effect of different concentrations of heparin on the fluorescence spectra of the QDs–Ru system was shown in Fig. 2A. Because of electrostatic interactions and hydrogen bonding between heparin molecule and Ru complex, heparin could interact with Ru complex and remove the quencher (Ru complex) from the surface of QDs when it was added, the fluorescence of QDs could be recovered. When 112 nM heparin was added, 83.4% of the QDs fluorescence intensity recovered. The research in our laboratory through molecular docking indicated that the electrostatic interactions might contribute to the metal center of Ru complex getting close to heparin molecule, furthermore, the hydrogen bonding could further induce heparin to closely “catch” the main ligand of Ru complex and the phz nitrogens was fully protected (Cheng et al., 2013). The binding constant of [Ru(phen)2(dppz-idzo)]2 þ for heparin was calculated to be 2.6 106 (Cheng et al. 2013). From the results shown in Fig. 2B, a linear relationship (I550 ¼ 1062.9þ 64.4 [heparin], R2 ¼0.991) of the heparin concentration with the fluorescence emission intensity was observed in the concentration range of 21–77 nM. LOD for heparin was 0.38 nM, based on 3 S0/ S, where S0 is the standard deviation of background and S is the sensitivity.
Y. Cao et al. / Biosensors and Bioelectronics 55 (2014) 174–179
177
Fig. 1. (A) Changes in the fluorescence spectra of TGA-capped CdTe QDs (88 nM) in 10 mM Tris–HCl buffer (pH 7.4) with increasing concentration of Ru(phen)2(dppzidzo)]2 þ (from top to bottom: 0, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2200 and 2400 nM); (B) plots of the fluorescence intensity of CdTe QDs against the concentration of Ru complex; (C) fluorescence intensity of the QDs–Ru complex in different buffer solutions including H2O, AC–NH4, HEPES, MOPS-NH2OH, PBS, Tris–AC, Tris–HCl buffer (pH 7.4) in the presence of 56 nM heparin; (D) plots of the fluorescence intensity the QDs–Ru complex in the presence of 56 nM heparin in 10 mM Tris–HCl buffer against pH values. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.3. Detection in fetal bovine serum (FBS) In order to demonstrate the practical application of this fluorescence assay for heparin detection in biological media, 1% fetal bovine serum (FBS) in 10 mM Tris–HCl buffer (pH 7.4) was used as the medium for further investigations. Fetal bovine serum contains proteins such as albumin, globulin and fibronectin and various anions including carboxylate ions. As shown in Fig. 2C, the fluorescence of QDs–Ru system gradually increased with successive addition of heparin in the diluted serum buffer solution. The saturation of the emission occurred at [Hep] E112 nM. The linear relationship between fluorescence intensity and heparin concentrations (I550 ¼ 880.183þ 35.8 [heparin], R2 ¼0.996) is 35–98 nM in 10 mM Tris–HCl buffer containing 1% FBS (pH 7.4) (Fig. 2D), and LOD is 0.68 nM. In addition, the fluorescence recovery of QDs–Ru by heparin in the diluted serum buffer solution is lower than that in Tris–HCl buffer. Something in FBS such as albumin, globulin, fibronectin or various anions may affect the fluorescence intensity of QDs, but it has no effect on detecting heparin in biological systems. Therefore, QDs–Ru complex is a rather promising fluorescence probe for the detection of heparin in biological systems.
triphosphates (ATP), uridine monophosphate (UMP), bovine serum albumin (BSA), chondroitin sulfate (Chs), hyaluronic acid (Hya) (Fig. S5). As we known, Chs and Hya are very similar to heparin in molecular structure. As shown in Fig. 2E, only heparin led to the increasing of QDs–Ru's fluorescence and the fluorescence intensity ranked as heparin c Chs 4Hya, implying that the conformation of the sugar dimer and electrostatic interactions play a dominant role in binding with Ru complex. These results demonstrate that QDs– Ru display good selectivity towards heparin over other similar biomolecules in this fluorescence assay in aqueous solution. This remarkable selectivity might be due to the structural compatibility between Ru complex and heparin. The research in our laboratory through molecular docking indicated that the sulphate and carboxylate groups attached on both sides of the heparin unit can closely “catch” one molecule of Ru complex, resulting in excimer emission, which differentiates heparin from similar biological entities, such as Chs and Hya (Cheng et al., 2013). Then, this detection selectivity was visualized under a 365 nm UV lamp (Fig. S6). Obviously, only heparin led to the change of the solution's color (Fig. 2E). Thus, the detection selectivity could be visualized with the naked eye under a 365 nm UV lamp.
3.4. Selectivity of fluorescence assay 4. Conclusions The selectivity of QDs–Ru based system was assessed by measuring the fluorescence intensity of the system in the presence of a series of biomolecules, including glucose, glycine, adenosine
In summary, we constructed a simple, ultrasensitive and selective fluorescent method for the detection of heparin by using the CdTe
178
Y. Cao et al. / Biosensors and Bioelectronics 55 (2014) 174–179
Fig. 2. (A) Fluorescence spectra of the QDs–Ru complex in 10 mM Tris–HCl buffer (pH 7.4) in the presence of different concentrations of heparin (from bottom to top: 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 98, 112, 126, 140, 154 and 168 nM); (B) plots of the fluorescence intensity in 10 mM Tris–HCl buffer (pH 7.4) against the concentration of heparin; (C) fluorescence spectra of the QDs–Ru complex in 10 mM Tris–HCl buffer containing 1% FBS (pH 7.4) in the presence of different concentrations of heparin (from bottom to top: 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 98, 112, 126, 140, 154 and 168 nM); (D) plots of the fluorescence intensity in 10 mM Tris–HCl buffer containing 1% FBS (pH 7.4) against the concentration of heparin; (E) fluorescence intensities of the QDs–Ru complex in 10 mM Tris–HCl buffer (pH 7.4) in the presence of different biomolecules. The fluorescence intensities were recorded at 550 nm. The concentration of heparin is 100 nM; concentrations of the other biomolecules are 1 μM. Excitation: 365 nm; Inset: photographs of the sensor in the presence of various biomolecules under a 365 nm UV lamp.
Quantum dots and a functional ruthenium polypyridyl complex [Ru(phen)2(dppz-idzo)]2 þ . This method possesses several superiorities: (1) The sensitivity is much higher and LOD (0.38 nM) is much lower than the reported “switch on” sensor for heparin based on [Ru (phen)2(dppz-idzo)]2þ alone (LOD is 0.011 U mL 1, or 10.5 nM calculated according to the heparin molecular mass of 6000 g mol 1) (Cheng et al., 2013) and other reported methods (Kuo and Tseng, 2013). (2) Utilizing QDs fluorescence, we detected heparin with the naked eye successfully. (3) Our assay is very convenient and can readily be used to detect heparin immediately without other treatments and can be used in biological systems. To the best of our
knowledge, this work represents the first example based on QDs and complex for heparin detection and it will provide potential applications in biological and medical fields.
Acknowledgements This work was supported by the National Science of Foundation of China (no. 81171646, no. 31170776, no. 20871094 and no. 20901060) and the Fundamental Research Funds for the Central Universities.
Y. Cao et al. / Biosensors and Bioelectronics 55 (2014) 174–179
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.12.009. References Algar, W.R., Susumu, K., Delehanty, J.B., Medintz, I.L., 2011. Anal. Chem. 83 (23), 8826–8837. Bowers, J., Ferguson, J.J., 1994. Clin. Cardiol. 17 (7), 357–361. Bromfield, S.M., Barnard, A., Posocco, P., Fermeglia, M., Pricl, S., Smith, D.K., 2013. J. Am. Chem. Soc. 135 (8), 2911–2914. Capila, I., Linhardt, R.J., 2002a. Angew. Chem. 114, 426–450. Capila, I., Linhardt, R.J., 2002b. Angew. Chem. Int. Ed. 41, 390–412. Chen, Z., Ren, X., Meng, X., Tan, L., Chen, D., Tang, F., 2013. Biosens. Bioelectron. 44, 204–209. Cheng, T., Lin, T., Wu, T., Chang, H., 2001. Anal. Chim. Acta 432 (1), 101–111. Cheng, T.T., Yao, J.L., Gao, X., Sun, W., Shi, S., Yao, T.M., 2013. Analyst 138 (12), 3483–3489. Friedman, A.E., Chambron, J.C., Sauvage, J.P., Turro, N.J., Barton, J.K., 1990. J. Am. Chem. Soc. 11, 4960–4962. Fu, X., Chen, L., Li, J., 2012a. Analyst 137 (16), 3653–3658. Fu, X., Chen, L., Li, J., Lin, M., You, H., Wang, W., 2012b. Biosens. Bioelectron. 34 (1), 227–231. Girolami, B., Girolami, A., 2006. Semin. Thromb. Hemost. 32 (8), 803–809. Guo, Z., Hao, T., Du, S., Chen, B., Wang, Z., Li, X., Wang, S., 2013. Biosens. Bioelectron. 44, 101–107. Jenkins, Y., Friedman, A.E., Turro, N.J., Barton, J.K., 1992. Biochemistry 31, 10809–10816. Kuo, C., Tseng, W., 2013. Chem. Commun. 49 (41), 4607–4609. Langmaier, J., Samcová, E., Samec, Z., 2007. Anal. Chem. 79 (7), 2892–2900. Li, M., Zhou, X., Guo, S., Wu, N., 2013. Biosens. Bioelectron. 43, 69–74. Patel, R.P., Narkowicz, C., Jacobson, G.A., 2009. Anal. Biochem. 387 (1), 113–121.
179
Patil, S.R., Mote, U.S., Patil, S.R., Kolekar, G.B., 2009. Bull. Korean Chem. Soc. 30 (12), 3034–3038. Peng, H., Zhang, L., Kjällman, T.H.M., Soeller, C., 2007. J. Am. Chem. Soc. 129 (11), 3048–3049. Rozenberg, G.I., Espada, J., De, C.L.L., Eijan, A.M., Calvo, J.C., Bertolesi, G.E., 2001. Electrophoresis 22, 3–11. Shi, S., Lv, C., Gao, X., Zhao, J., Yao, J., Sun, W., Huang, H., Yao, T., Ji, L., 2012a. Inorg. Chem. Commun. 24, 212–215. Shi, S., Zhao, J., Gao, X., Lv, C., Yang, L., Hao, J., Huang, H., Yao, J., Sun, W., Yao, T., Ji, L., 2012b. Dalton Trans. 41, 5789–5793. Shi, S., Zhao, J., Geng, X., Yao, T., Huang, H., Liu, T., Zheng, L., Li, Z., Yang, D., Ji, L., 2010a. Dalton Trans. 39, 2490–2493. Shi, S., Geng, X., Zhao, J., Yao, T., Wang, C., Yang, D., Zheng, L., Ji, L., 2010b. Biochimie 92, 370–377. Shi, S., Huang, H., Gao, X., Yao, J., Lv, C., Zhao, J., Sun, W., Yao, T., Ji, L., 2013a. J. Inorg. Biochem. 121, 19–27. Shi, S., Wang, X., Sun, W., Wang, X., Yao, T., Ji, L., 2013b. Methods 64 (3), 305–314. Shriver, Z., Raman, R., Venkataraman, G., Drummond, K., Turnbull, J., Toida, T., Linhardt, R., Biemann, K., Sasisekharan, R., 2000. Proc. Nat. Acad. Sci. U.S.A 97 (19), 10359–10364. Smith, A.M., Duan, H., Mohs, A.M., Nie, S., 2008. Adv. Drug. Delivery Rev. 60 (11), 1226–1240. Sun, W., Shi, S., Yao, T., 2011. Anal. Methods 3 (11), 2472–2474. Sun, W., Yao, J., Yao, T., Shi, S., 2013. Analyst 138 (2), 421–424. Sun, W., Yao, T., Shi, S., 2012. Analyst 137 (7), 1550–1552. Sykora, M., Petruska, M.A., Alstrum-Acevedo, J., Bezel, I., Meyer, T.J., Klimov, V.I., 2006. J. Am. Chem. Soc. 128 (31), 9984–9985. Szelke, H., Harenberg, J., Krämer, R., 2009. Thromb. Haemost. 102, 859–864. Wen, S., Zheng, F., Shen, M., Shi, X., 2013. Colloids Surf., A 419, 80–86. Yan, H., Wang, H.F., 2011. Anal. Chem. 83 (22), 8589–8595. Yao, J.L., Gao, X., Sun, W., Fan, X.Z., Shi, S., Yao, T.M., 2012. Inorg. Chem. 51 (23), 12591–12593. Yao, J.L., Gao, X., Sun, W., Shi, S., Yao, T.M., 2013. Dalton Trans. 42 (16), 5661–5672. Yu, W.W., Qu, L., Guo, W., Peng, X., 2003. Chem. Mater. 15 (14), 2854–2860.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Ultrasensitive fluorescence detection of heparin based on quantum dots and a functional ruthenium polypyridyl complex Yanlin Cao, Shuo Shi n, Linlin Wang, Junliang Yao, Tianming Yao n Department of Chemistry, Tongji University, 1239 Siping Road, Shanghai 200092, China
art ic l e i nf o
a b s t r a c t
Article history: Received 6 September 2013 Received in revised form 21 November 2013 Accepted 2 December 2013 Available online 12 December 2013
A new strategy for the detection of heparin is developed by utilizing quantum dots (QDs) and a functional ruthenium polypyridyl complex [Ru(phen)2(dppz-idzo)]2 þ (phen ¼ 1,10-phenanthroline, dppz-idzo ¼dipyrido-[3,2-a:20 ,30 -c] phenazine-imidazolone). The emission of CdTe QDs is found to be quenched by Ru complex due to electron transfer. Upon addition of the polyanionic heparin, it could remove the quencher (Ru complex) from the surface of QDs owing to the electrostatic and/or hydrogen bonding interactions between heparin and Ru complex, which led to significant fluorescence recovery of CdTe QDs. The fluorescence intensity enhanced with the increase of heparin and a linear relationship was observed in the range of 21–77 nM for heparin detection in buffer solution and the limit of detection (LOD) is 0.38 nM. Moreover, the strategy was successfully applied to detect heparin as low as 0.68 nM with a linear range of 35–98 nM in fetal bovine serum samples. The selectivity results of the fluorescence assay revealed that our system displayed excellent fluorescence selectivity towards heparin over its analogues, such as chondroitin 4-sulfate (Chs) or hyaluronic acid (Hya). This fluorescence “switch on” assay for heparin is label-free, convenient, sensitive and selective, which can be used to detect heparin in biological systems even with the naked eyes. & 2013 Elsevier B.V. All rights reserved.
Keywords: Quantum dots Ru complex Fluorescence Heparin Fetal bovine serum
1. Introduction Heparin is a highly negatively charged sulfated linear polymeric glycosaminoglycan (Capila and Linhardt, 2002a, 2002b), it has been clinically used in different forms in anticoagulation treatment during cardiopulmonary surgery and in emergency deep venous thrombosis conditions (Shriver et al., 2000). While, the overdose of heparin often causes potentially fatal bleeding complication such as hemorrhages and thrombocytopenia (Girolami and Girolami, 2006). So it is of crucial importance to monitor closely heparin levels for the sake of health. So far many efforts have been devoted to the detection and quantification of heparin. The conventional methods for heparin detection include activated partial thromboplastin time (Cheng et al., 2001), activated clotting time (Bowers and Ferguson, 1994), potentiometric assays (Langmaier et al., 2007) and ion pair high performance liquid chromatography (Patel et al., 2009). However, these methods exist some problems such as the lack of specificity and potential interference from other factors. Recently, more researches have focused on the direct detection of heparin by binding to sensing device or probe, such as PEI/AuNPs as a probe for the colorimetric detection of heparin (Wen et al., 2013), GO quenching the color of self-assembly
n
Corresponding authors. Tel./fax: þ86 21 65983292. E-mail addresses: [email protected] (S. Shi), [email protected] (T. Yao).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.12.009
gold nanorods (Fu et al., 2012b) and gold nanoparticles (Fu et al., 2012a), target-induced self-assembly of polyethyleneimine capped Mn-doped ZnS quantum dots (Yan and Wang, 2011), adenosinebased molecular beacons as light-up probes (Kuo and Tseng, 2013), mallard blue as a sensor via simple UV–vis measurements (Bromfield et al., 2013), norfloxacin–cerium complex as a spectofluorimetric sensor (Patil et al., 2009) and so on. Among these methods, fluorescent chemosensors received intense attention recently, however, some of them have limitations including poor sensitivity and selectivity. Heretofore, ultrasensitive fluorescence “switch-on” sensors for heparin, which have good selectivity, and can be used for direct monitoring of heparin in serum, still remain rare. Therefore, there is still a high demand for a convenient, sensitive and rapid method for recognizing and sensing of heparin. In addition, ruthenium polypyridyl complexes have been widely investigated because of their prominent DNA-binding properties (Friedman et al., 1990; Jenkins et al., 1992). Our laboratory has also studied the binding behaviors of many ruthenium complexes with various DNA structures (Shi et al., 2010a, 2010b, 2012a, 2012b, 2013a, 2013b; Sun et al., 2011). However, few has been reported heretofore about the interaction between Ru(II) complexes and heparin. Rozenberg first used a ruthenium complex [Ru(2,20 -bipyridine)3]2 þ for the detection of heparin in gels by fluorescent staining (after electrophoresis, isolation, incubation and washing) (Rozenberg et al., 2001). But this complex does not respond to heparin in buffered solution or serum containing samples. Later, Szelke et al. (2009) attached
Y. Cao et al. / Biosensors and Bioelectronics 55 (2014) 174–179
cationic residues to the bipyridine ligands and got a polycationic fluorescent ruthenium complex. They succeeded to detect heparin in buffer solution or serum. However, this “switch-off” detection suffered from the low sensitivity and specificity. In our recent study, a new strategy for heparin detection and quantification in biological media, such as fetal bovine serum (FBS), was developed by monitoring the emission change of a ruthenium polypyridyl complex [Ru (phen)2(dppz-idzo)]2 þ in buffer solutions (Cheng et al., 2013). [Ru (phen)2dppz-idzo]2 þ was almost non-emissive in aqueous solution due to the hydrogen bonding between the solvent and the phz (phenazine) nitrogens on the main ligand (dppz-idzo) (Yao et al., 2013). However, in the presence of heparin, the complex was supposed to interact with the sulfanilamido, sulfate and carboxylate groups on the pyranosyluronic acid unit in heparin through electrostatic and/or hydrogen bonding interactions and got protected from water, resulting in excimer emission of complex [Ru(phen)2dppzidzo]2 þ . Thus, our complex could be employed for the fluorescence “switch on” detection of heparin. However, the sensitivity of this method was unsatisfied, the limit of detection (0.011 U mL 1, or 10.5 nM calculated according to the heparin molecular mass of 6000 g mol 1) was high. In order to solve these problems, we designed another strategy to detect heparin based on CdTe quantum dots and Ru complex [Ru(phen)2(dppz-idzo)]2 þ . Quantum dots (QDs), one of the most attractive nanometer-sized luminescent semiconductor crystals, have many advantages over organic dyes such as excellent photostability, high quantum yield, tunable emission wavelength, and high photobleaching threshold (Algar et al., 2011). In addition, the large surface of QDs is favorable for attaching variable ligands and thus getting controllable properties (Smith et al., 2008). Up to now, QDs have been widely used as novel luminescence indicators of different biological processes and bioanalysis in recent years (Chen et al., 2013; Guo et al., 2013; Li et al., 2013). The interactions between QDs and Ru polypyridine complexes in solution have been reported (Sykora et al., 2006). The emission of QDs could be quenched by Ru complexes and the possible mechanism for QDs emission quenching is the photo induced electron transfer (Sun et al., 2013). In our laboratory, we have proposed label-free, aptamer-based biosensors for the selective detection of thrombin (Sun et al., 2012) and silver ions (Sun et al., 2013) by using a molecule “light switch” Ru polypyridine complex [Ru(bpy)2(dppz)]2 þ and QDs. Herein, we designed a label free, sensitive and selective luminescence sensor for heparin based on CdTe quantum dots and Ru complex [Ru(phen)2(dppz-idzo)]2 þ (Scheme 1). Upon addition of the Ru complex, the positively charged Ru complex and negatively charged water-soluble CdTe quantum dots form an anionic conjugate due to their electrostatic attraction in aqueous solution. This static association between the Ru complex and QDs induces ultrafast photoinduced electron transfer from QDs to the electron acceptor (the Ru complex), preventing the normal recombination of the electron and the hole in QDs, and results in a decrease of the fluorescent intensity of QDs. When added into the mixture, heparin could remove Ru complex (the quencher) from the surface of QDs due to its high avidity for Ru complex, and the fluorescence of QDs could be recovered. This sensing platform provides an ultrasensitive, simple, and selective method for the detection of heparin.
2. Materials and methods 2.1. Materials Heparin, NaBH4, tellurium powder, CdCl2, thioglycolic acid and NaOH was purchased from Sigma-Aldrich. Glucose, glycine, adenosine triphosphates (ATP), uridine monophosphate (UMP), bovine serum albumin (BSA), chondroitin sulfate (Chs), hyaluronic
175
acid (Hya), and fetal bovine serum (FBS) were obtained from Genestar, Shanghai, China. [Ru(phen)2dppz-idzo)]2 þ was synthesized by our laboratory according to the literature (Yao et al., 2012). Heparin sodium salt was got from porcine intestinal mucosa and 1 μM heparin corresponded to 6 μg/mL (Molecular mass: 6.0 kDa). Stock solutions of heparin, Chs, and Hya in water were prepared and stored at 4 1C before use. Fetal bovine serum was stored at 20 1C and was brought to room temperature when used. Tris–HCl buffer solution (10 mM Tris, pH 7.4) was used in all of the experiment. 2.2. Instrumentation The fluorescence spectra were recorded at room temperature on an F-7000 fluorescence spectrophotometer (Hitachi) with a quartz cell (1 mm). Both the excitation and emission slit widths were fixed at 10 nm. Lambda Bio 40 UV/Vis Spectrophotometer (Perkin-Elmer, USA) was used to quantify the QDs. 2.3. Preparation of CdTe QDs The synthesis of CdTe QDs was performed according to the reference with some modification (Peng et al., 2007) (In electronic supplementary information). The UV–vis spectrum of TGA (thioglycolic acid)-capped CdTe QDs was obtained (Fig. S1). The sizes and concentrations of QDs can be estimated from the adsorption peaks in UV–vis spectrum (Yu et al., 2003). 2.4. Optimization of the sensor For the detection of the quenching behavior of [Ru(phen)2(dppzidzo)]2 þ on the fluorescence of QDs, 10 μL CdTe QDs and various amounts of [Ru(phen)2(dppz-idzo)]2 þ were incubated in 10 mM Tris–HCl buffer (pH 7.4) at room temperature for 5 min in a 1.5 mL Eppendorf tube. The final concentration of CdTe QDs was 88 nM. Then the fluorescence spectra were measured in a 2 mL quartz cuvette at room temperature. In order to obtain a highly sensitive response for the detection of heparin, the optimization of the conditions including buffer solution and pH value were carried out in our experiment. We selected H2O, AC–NH4, HEPES, MOPS-NH2OH, PBS, Tris–AC, Tris– HCl as buffer solutions. A sample of 10 μL CdTe QDs and 70 μL [Ru(phen)2(dppz-idzo)]2 þ (20 μM) were incubated for 5 min in different buffer solutions. Next, 80 μL heparin (0.7 μM) was mixed with this solution and incubated at room temperature for another 5 min. The resulting solutions were studied by fluorescent spectroscopy at room temperature with excitation at 365 nm, both the excitation and emission slit widths were 10 nm. In the same way, the optimization of pH value was carried out in Tris–HCl buffer solution with different pH values. 2.5. Fluorescence detection of heparin in buffer solution For the fluorescence assay of heparin, 10 μL CdTe QDs and 70 μL [Ru(phen)2(dppz-idzo)]2 þ (20 μM) were incubated for 5 min in 10 mM Tris–HCl buffer (pH 7.4). Next, different concentration of heparin was mixed with this solution and incubated at room temperature for another 5 min. The final concentration of Ru complex was 1.4 μM, CdTe QDs was 88 nM. Then the fluorescence spectra were measured. 2.6. Analysis of heparin in fetal bovine serum All fluorescence assays were carried out in biological media, 1% fetal bovine serum (FBS) in 10 mM Tris–HCl buffer.
176
Y. Cao et al. / Biosensors and Bioelectronics 55 (2014) 174–179
Scheme 1. Schematic description of fluorescence detection of heparin by utilizing CdTe quantum dots and Ru complex [Ru(phen)2(dppz-idzo)]2 þ and chemical structures of Chs, Hya, heparin.
2.7. Sensor selectivity investigation In the selectivity experiment, 10 μL CdTe QDs and 70 μL [Ru (phen)2(dppz-idzo)]2 þ (20 μM) were incubated for 5 min in 10 mM Tris–HCl buffer (pH 7.4). Next, a series of biomolecules, including glucose, glycine, adenosine triphosphates (ATP), uridine monophosphate (UMP), bovine serum albumin (BSA), chondroitin sulfate (Chs), hyaluronic acid (Hya) were mixed with this solution and incubated at room temperature for another 5 min. The concentration of heparin was 100 nM, the other biomolecules was 1 μM, CdTe QDs was 88 nM and Ru complex was 1.4 μM. The resulting solutions were studied by fluorescent spectroscopy at room temperature with excitation at 365 nm, both the excitation and emission slit width were 10 nm.
3. Results and discussion 3.1. Optimization of the sensor To investigate the quenching behavior of [Ru(phen)2(dppzidzo)]2 þ on the fluorescence of CdTe QDs, the fluorescence signals toward different concentrations of Ru complex were measured in 10 mM Tris–HCl buffer (pH 7.4). The fluorescence intensity decreased with increasing of Ru complex and the emission intensity plotted against the concentration of Ru complex were shown in Fig. 1A and Fig. 1B, respectively. The free QDs show strong fluorescence emission in Tris–HCl buffer. When 1.4 μM Ru complex was added, the fluorescence intensity of QDs (88 nM) could decrease to 0.57%, indicating Ru complex could quench the fluorescence of QDs by electronic interactions that occurred upon attachment. We figured out that the binding constant of Ru complex for QDs was 5.53 105 (In electronic supplementary information). The high quenching efficiency would lead to a high signal-to-back ground ratio and good sensitivity for target detection. In our later experiments for heparin detection, 1.4 μM Ru complex was selected to ensure high quenching efficiency. QDs and Ru complex could interact and then formed a fluorescencequenched complex which was used for analytical purposes, named QDs–Ru, hereafter. Buffer solution and pH values can not only affect the fluorescence intensity of QDs, but also affect the interaction between [Ru (phen)2(dppz-idzo)]2þ and heparin. For this reason, it is important to select an appropriate buffer and pH value for assay of heparin. Here,
six kind buffer solutions including AC–NH4, HEPES, MOPS–NH2OH, PBS, Tris–AC, Tris–HCl were investigated (Fig. S3). The concentration of each buffer was 10 mM and the pH was 7.4. The result (Fig. 1C) shown that the fluorescence intensity in Tris–HCl was higher than that in other five buffers. Thus, Tris–HCl was selected in this study for all other experiments. As for the pH value, the effect of pH within a range from 3.5 to 8.5 was investigated (Fig. S4). As shown in Fig. 1D, the fluorescence intensity increased gradually with the pH ranging from 4.0 to 8.5, but the increase from 7.5 to 8.5 was not so obvious. The pH value in the body is 7.35–7.45 and pH 7.4 is a mild condition, therefore, pH 7.4 was selected in this study.
3.2. The analytical performance of the sensor The free [Ru(phen)2(dppz-idzo)]2 þ has no fluorescence, but it can emit fluorescence at 630 nm after binding to heparin (Fig. S2). However, the fluorescence intensity is much lower than that of QDs and the fluorescence emission has a red shift in our system (Fig. 1). Thus, we mainly focused on the fluorescence intensity of QDs in our experiments. To investigate our strategy for the detection of heparin, the emission of QDs–Ru as a function of heparin concentration was measured. The effect of different concentrations of heparin on the fluorescence spectra of the QDs–Ru system was shown in Fig. 2A. Because of electrostatic interactions and hydrogen bonding between heparin molecule and Ru complex, heparin could interact with Ru complex and remove the quencher (Ru complex) from the surface of QDs when it was added, the fluorescence of QDs could be recovered. When 112 nM heparin was added, 83.4% of the QDs fluorescence intensity recovered. The research in our laboratory through molecular docking indicated that the electrostatic interactions might contribute to the metal center of Ru complex getting close to heparin molecule, furthermore, the hydrogen bonding could further induce heparin to closely “catch” the main ligand of Ru complex and the phz nitrogens was fully protected (Cheng et al., 2013). The binding constant of [Ru(phen)2(dppz-idzo)]2 þ for heparin was calculated to be 2.6 106 (Cheng et al. 2013). From the results shown in Fig. 2B, a linear relationship (I550 ¼ 1062.9þ 64.4 [heparin], R2 ¼0.991) of the heparin concentration with the fluorescence emission intensity was observed in the concentration range of 21–77 nM. LOD for heparin was 0.38 nM, based on 3 S0/ S, where S0 is the standard deviation of background and S is the sensitivity.
Y. Cao et al. / Biosensors and Bioelectronics 55 (2014) 174–179
177
Fig. 1. (A) Changes in the fluorescence spectra of TGA-capped CdTe QDs (88 nM) in 10 mM Tris–HCl buffer (pH 7.4) with increasing concentration of Ru(phen)2(dppzidzo)]2 þ (from top to bottom: 0, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2200 and 2400 nM); (B) plots of the fluorescence intensity of CdTe QDs against the concentration of Ru complex; (C) fluorescence intensity of the QDs–Ru complex in different buffer solutions including H2O, AC–NH4, HEPES, MOPS-NH2OH, PBS, Tris–AC, Tris–HCl buffer (pH 7.4) in the presence of 56 nM heparin; (D) plots of the fluorescence intensity the QDs–Ru complex in the presence of 56 nM heparin in 10 mM Tris–HCl buffer against pH values. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.3. Detection in fetal bovine serum (FBS) In order to demonstrate the practical application of this fluorescence assay for heparin detection in biological media, 1% fetal bovine serum (FBS) in 10 mM Tris–HCl buffer (pH 7.4) was used as the medium for further investigations. Fetal bovine serum contains proteins such as albumin, globulin and fibronectin and various anions including carboxylate ions. As shown in Fig. 2C, the fluorescence of QDs–Ru system gradually increased with successive addition of heparin in the diluted serum buffer solution. The saturation of the emission occurred at [Hep] E112 nM. The linear relationship between fluorescence intensity and heparin concentrations (I550 ¼ 880.183þ 35.8 [heparin], R2 ¼0.996) is 35–98 nM in 10 mM Tris–HCl buffer containing 1% FBS (pH 7.4) (Fig. 2D), and LOD is 0.68 nM. In addition, the fluorescence recovery of QDs–Ru by heparin in the diluted serum buffer solution is lower than that in Tris–HCl buffer. Something in FBS such as albumin, globulin, fibronectin or various anions may affect the fluorescence intensity of QDs, but it has no effect on detecting heparin in biological systems. Therefore, QDs–Ru complex is a rather promising fluorescence probe for the detection of heparin in biological systems.
triphosphates (ATP), uridine monophosphate (UMP), bovine serum albumin (BSA), chondroitin sulfate (Chs), hyaluronic acid (Hya) (Fig. S5). As we known, Chs and Hya are very similar to heparin in molecular structure. As shown in Fig. 2E, only heparin led to the increasing of QDs–Ru's fluorescence and the fluorescence intensity ranked as heparin c Chs 4Hya, implying that the conformation of the sugar dimer and electrostatic interactions play a dominant role in binding with Ru complex. These results demonstrate that QDs– Ru display good selectivity towards heparin over other similar biomolecules in this fluorescence assay in aqueous solution. This remarkable selectivity might be due to the structural compatibility between Ru complex and heparin. The research in our laboratory through molecular docking indicated that the sulphate and carboxylate groups attached on both sides of the heparin unit can closely “catch” one molecule of Ru complex, resulting in excimer emission, which differentiates heparin from similar biological entities, such as Chs and Hya (Cheng et al., 2013). Then, this detection selectivity was visualized under a 365 nm UV lamp (Fig. S6). Obviously, only heparin led to the change of the solution's color (Fig. 2E). Thus, the detection selectivity could be visualized with the naked eye under a 365 nm UV lamp.
3.4. Selectivity of fluorescence assay 4. Conclusions The selectivity of QDs–Ru based system was assessed by measuring the fluorescence intensity of the system in the presence of a series of biomolecules, including glucose, glycine, adenosine
In summary, we constructed a simple, ultrasensitive and selective fluorescent method for the detection of heparin by using the CdTe
178
Y. Cao et al. / Biosensors and Bioelectronics 55 (2014) 174–179
Fig. 2. (A) Fluorescence spectra of the QDs–Ru complex in 10 mM Tris–HCl buffer (pH 7.4) in the presence of different concentrations of heparin (from bottom to top: 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 98, 112, 126, 140, 154 and 168 nM); (B) plots of the fluorescence intensity in 10 mM Tris–HCl buffer (pH 7.4) against the concentration of heparin; (C) fluorescence spectra of the QDs–Ru complex in 10 mM Tris–HCl buffer containing 1% FBS (pH 7.4) in the presence of different concentrations of heparin (from bottom to top: 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 98, 112, 126, 140, 154 and 168 nM); (D) plots of the fluorescence intensity in 10 mM Tris–HCl buffer containing 1% FBS (pH 7.4) against the concentration of heparin; (E) fluorescence intensities of the QDs–Ru complex in 10 mM Tris–HCl buffer (pH 7.4) in the presence of different biomolecules. The fluorescence intensities were recorded at 550 nm. The concentration of heparin is 100 nM; concentrations of the other biomolecules are 1 μM. Excitation: 365 nm; Inset: photographs of the sensor in the presence of various biomolecules under a 365 nm UV lamp.
Quantum dots and a functional ruthenium polypyridyl complex [Ru(phen)2(dppz-idzo)]2 þ . This method possesses several superiorities: (1) The sensitivity is much higher and LOD (0.38 nM) is much lower than the reported “switch on” sensor for heparin based on [Ru (phen)2(dppz-idzo)]2þ alone (LOD is 0.011 U mL 1, or 10.5 nM calculated according to the heparin molecular mass of 6000 g mol 1) (Cheng et al., 2013) and other reported methods (Kuo and Tseng, 2013). (2) Utilizing QDs fluorescence, we detected heparin with the naked eye successfully. (3) Our assay is very convenient and can readily be used to detect heparin immediately without other treatments and can be used in biological systems. To the best of our
knowledge, this work represents the first example based on QDs and complex for heparin detection and it will provide potential applications in biological and medical fields.
Acknowledgements This work was supported by the National Science of Foundation of China (no. 81171646, no. 31170776, no. 20871094 and no. 20901060) and the Fundamental Research Funds for the Central Universities.
Y. Cao et al. / Biosensors and Bioelectronics 55 (2014) 174–179
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.12.009. References Algar, W.R., Susumu, K., Delehanty, J.B., Medintz, I.L., 2011. Anal. Chem. 83 (23), 8826–8837. Bowers, J., Ferguson, J.J., 1994. Clin. Cardiol. 17 (7), 357–361. Bromfield, S.M., Barnard, A., Posocco, P., Fermeglia, M., Pricl, S., Smith, D.K., 2013. J. Am. Chem. Soc. 135 (8), 2911–2914. Capila, I., Linhardt, R.J., 2002a. Angew. Chem. 114, 426–450. Capila, I., Linhardt, R.J., 2002b. Angew. Chem. Int. Ed. 41, 390–412. Chen, Z., Ren, X., Meng, X., Tan, L., Chen, D., Tang, F., 2013. Biosens. Bioelectron. 44, 204–209. Cheng, T., Lin, T., Wu, T., Chang, H., 2001. Anal. Chim. Acta 432 (1), 101–111. Cheng, T.T., Yao, J.L., Gao, X., Sun, W., Shi, S., Yao, T.M., 2013. Analyst 138 (12), 3483–3489. Friedman, A.E., Chambron, J.C., Sauvage, J.P., Turro, N.J., Barton, J.K., 1990. J. Am. Chem. Soc. 11, 4960–4962. Fu, X., Chen, L., Li, J., 2012a. Analyst 137 (16), 3653–3658. Fu, X., Chen, L., Li, J., Lin, M., You, H., Wang, W., 2012b. Biosens. Bioelectron. 34 (1), 227–231. Girolami, B., Girolami, A., 2006. Semin. Thromb. Hemost. 32 (8), 803–809. Guo, Z., Hao, T., Du, S., Chen, B., Wang, Z., Li, X., Wang, S., 2013. Biosens. Bioelectron. 44, 101–107. Jenkins, Y., Friedman, A.E., Turro, N.J., Barton, J.K., 1992. Biochemistry 31, 10809–10816. Kuo, C., Tseng, W., 2013. Chem. Commun. 49 (41), 4607–4609. Langmaier, J., Samcová, E., Samec, Z., 2007. Anal. Chem. 79 (7), 2892–2900. Li, M., Zhou, X., Guo, S., Wu, N., 2013. Biosens. Bioelectron. 43, 69–74. Patel, R.P., Narkowicz, C., Jacobson, G.A., 2009. Anal. Biochem. 387 (1), 113–121.
179
Patil, S.R., Mote, U.S., Patil, S.R., Kolekar, G.B., 2009. Bull. Korean Chem. Soc. 30 (12), 3034–3038. Peng, H., Zhang, L., Kjällman, T.H.M., Soeller, C., 2007. J. Am. Chem. Soc. 129 (11), 3048–3049. Rozenberg, G.I., Espada, J., De, C.L.L., Eijan, A.M., Calvo, J.C., Bertolesi, G.E., 2001. Electrophoresis 22, 3–11. Shi, S., Lv, C., Gao, X., Zhao, J., Yao, J., Sun, W., Huang, H., Yao, T., Ji, L., 2012a. Inorg. Chem. Commun. 24, 212–215. Shi, S., Zhao, J., Gao, X., Lv, C., Yang, L., Hao, J., Huang, H., Yao, J., Sun, W., Yao, T., Ji, L., 2012b. Dalton Trans. 41, 5789–5793. Shi, S., Zhao, J., Geng, X., Yao, T., Huang, H., Liu, T., Zheng, L., Li, Z., Yang, D., Ji, L., 2010a. Dalton Trans. 39, 2490–2493. Shi, S., Geng, X., Zhao, J., Yao, T., Wang, C., Yang, D., Zheng, L., Ji, L., 2010b. Biochimie 92, 370–377. Shi, S., Huang, H., Gao, X., Yao, J., Lv, C., Zhao, J., Sun, W., Yao, T., Ji, L., 2013a. J. Inorg. Biochem. 121, 19–27. Shi, S., Wang, X., Sun, W., Wang, X., Yao, T., Ji, L., 2013b. Methods 64 (3), 305–314. Shriver, Z., Raman, R., Venkataraman, G., Drummond, K., Turnbull, J., Toida, T., Linhardt, R., Biemann, K., Sasisekharan, R., 2000. Proc. Nat. Acad. Sci. U.S.A 97 (19), 10359–10364. Smith, A.M., Duan, H., Mohs, A.M., Nie, S., 2008. Adv. Drug. Delivery Rev. 60 (11), 1226–1240. Sun, W., Shi, S., Yao, T., 2011. Anal. Methods 3 (11), 2472–2474. Sun, W., Yao, J., Yao, T., Shi, S., 2013. Analyst 138 (2), 421–424. Sun, W., Yao, T., Shi, S., 2012. Analyst 137 (7), 1550–1552. Sykora, M., Petruska, M.A., Alstrum-Acevedo, J., Bezel, I., Meyer, T.J., Klimov, V.I., 2006. J. Am. Chem. Soc. 128 (31), 9984–9985. Szelke, H., Harenberg, J., Krämer, R., 2009. Thromb. Haemost. 102, 859–864. Wen, S., Zheng, F., Shen, M., Shi, X., 2013. Colloids Surf., A 419, 80–86. Yan, H., Wang, H.F., 2011. Anal. Chem. 83 (22), 8589–8595. Yao, J.L., Gao, X., Sun, W., Fan, X.Z., Shi, S., Yao, T.M., 2012. Inorg. Chem. 51 (23), 12591–12593. Yao, J.L., Gao, X., Sun, W., Shi, S., Yao, T.M., 2013. Dalton Trans. 42 (16), 5661–5672. Yu, W.W., Qu, L., Guo, W., Peng, X., 2003. Chem. Mater. 15 (14), 2854–2860.
