Cyclodextrin supramolecular inclusion-enhanced pyrene excimer switching for time-resolved fluorescence detection of biothiols in serum

Biosensors and Bioelectronics 68 (2015) 253–258

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Cyclodextrin supramolecular inclusion-enhanced pyrene excimer switching for time-resolved fluorescence detection of biothiols in serum Qier Zhang a,b, Ting Deng a,b,n, Jishan Li b, Weijian Xu b, Guoli Shen b, Ruqin Yu b a b

Institute of Applied Chemistry, School of Science, Central South University of Forestry and Technology, Changsha 410004, China State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China

art ic l e i nf o

a b s t r a c t

Article history: Received 17 October 2014 Received in revised form 27 December 2014 Accepted 2 January 2015 Available online 3 January 2015

We report here an efficient pyrene excimer signaling-based time-resolved fluorescent sensor for the measurement of biothiols (cysteine (Cys), homocysteine (Hcy), glutathione (GSH)) in human serum based on thymine–Hg2 þ –thymine (T–Hg2 þ –T) coordination chemistry and the inclusion interaction of cyclodextrin. The sensing mechanism of the approach is based on the competitive ligation of Hg2 þ ions by Hcy/Cys/GSH and T–T mismatches in a bis-pyrene-labeled DNA strand with the self-complementary 5′ and 3′ ends. The introduction of γ-cyclodextrin can provide cooperation for the molecular level space proximity of the two labeled pyrene molecules, moreover the hydrophobic cavity of γ-cyclodextrin can also offer protection for the pyrene dimer's emission from the quenching effect of environmental conditions and enhance the fluorescence intensity of the pyrene excimer. When the biothiols are not presented, the sensing ensemble is in the “off” state due to the long distance between the two labeled pyrene molecules resulted from the formation of a more stable T–Hg2 þ –T structure. While in the presence of biothiols, Hg2 þ interacts very strongly with thiol groups and the T–Hg2 þ –T structure is dehybridized, and then the pyrene excimer will be formed due to the self-complementary 5′ and 3′ ends of the DNA probe and the cooperation interaction of γ-cyclodextrin to pyrene dimer, thus resulting in switching the sensing ensemble to the “on” state. In the optimum conditions described, the linear concentration range of 1.0–100 μM with the limit of detection (LOD) of 0.36 μM for GSH was obtained. Moreover, due to the much longer lifetime of the pyrene excimer fluorescence than those of the ubiquitous endogenous fluorescent components, the time-resolved fluorescence technique has been successfully used for application in complicated biological samples. & 2015 Elsevier B.V. All rights reserved.

Keywords: Time-resolved fluorescence technique Pyrene excimer Thymine–Hg2 þ –thymine coordination chemistry Inclusion interaction Biothoils

1. Introduction Biothiols, including thiol-containing amino acids and peptides (glutathione (GSH), cysteine (Cys), homocysteine (Hcy)), as important structural or functional components of many proteins and enzymes play many crucial roles in biological systems (Wood et al., 2003; Dalton et al., 1999; Kanzok et al., 2000; Krauth-Siegel et al., 2005; Schirmer et al., 1995). Moreover, many researches indicated that the level of biothiols in plasma has been linked to lots of diseases for example cardiovascular and Alzheimer's disease (Refsum et al., 1989; Seshadri et al., 2002), AIDS (Staal et al., 1992), and can also be as an indicator for various medical conditions (Perlman et al., 1940; Saravanan et al., 1996; Droge and Holm, n Corresponding author at: Institute of Applied Chemistry, School of Science, Central South University of Forestry and Technology, Changsha 410004, China. Fax: þ86 731 85623353. E-mail address: [email protected] (T. Deng).

http://dx.doi.org/10.1016/j.bios.2015.01.004 0956-5663/& 2015 Elsevier B.V. All rights reserved.

1997; Goodman et al., 2000; Liu et al., 2000). Due to their important roles in biological systems, great attention has been paid to the assay of biothiols, such as the traditional chromatography or capillary electrophoresis methods (Shahrokhian, 2001; Zen et al., 2001), electrochemical voltammetry methods (Zhao et al., 2003; Tseng et al., 2006), fluorescent assay methods (Rusin et al., 2004; Tanaka et al., 2004; Wang et al., 2005; Zhang et al., 2007; Xu and Hepel, 2011), gold nanoparticle aggregation-based colorimetric methods and so on (Lee et al., 2008; Li and Li, 2009). Among these assay protocols for biothiols, fluorescence assays have received much attention for their high sensitivity and simplicity in operation. However, owing to the diffusion and natural fluorescence of various compounds or proteins in biological samples (Fu et al., 2007), these conventional fluorescent methods are mostly suffered from serious limitations of application in complicated biological samples (for example serums) due to the high natural fluorescent background. Therefore, the new fluorescent sensing strategies with the property of low biological auto-fluorescence, high

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selectivity, high sensitivity and excellent performance are still highly demanded. The limitations in traditional fluorescent methods motivated us to design the long lifetime fluorescent dye-employed strategies to realize the assay of biothiols in complicated biological samples through the use of time-resolved fluorescence technique. Generally, the auto-fluorescence lifetime of biological samples is less than 10 ns, so the background interference from ubiquitous endogenous fluorescent components could be reduced greatly when the long lifetime fluorescent dye is employed through the timeresolved fluorescence technique (De Haas et al., 1996; Bjartell et al., 1999; Connally et al., 2004; Weibel et al., 2004; Song et al., 2006; Lammers et al., 2009). In recent years, although a variety of organic molecules- or lanthanide ion luminescent complexesbased time-resolved fluorescence sensing strategies have been reported for various bio-targets such as metal ions (Gao et al., 2013), nucleic acids (Conlon et al., 2008; Karhunen et al., 2010; Krasnoperov et al., 2010) and proteins (Reichstein et al., 1988; Yang et al., 2005; Egashira et al., 2008; Ouyang et al., 2011), the timeresolved fluorescence-based sensing methods for biothiols assay in complicated biological samples has not been reported so far. Hence, considering the unique properties of the long lifetime fluorescent species or the time-resolved fluorescence technique and the importance of biothiols assay in complicated biological samples, exploration of long lifetime fluorescent species-employed sensing strategies for biothiols assay in complicated biological samples will have a fascinating prospect. Herein, we report the proof-of-principle of a thymine–Hg2 þ – thymine (T–Hg2 þ –T) coordination chemistry and cyclodextrin supermolecular inclusion-based pyrene excimer switching ensemble for time-resolved fluorescence assay of biothiols in serum (Scheme 1). Pyrene is a simple hydrocarbon aromatic molecule (Birks, 1979), and pyrene-based fluorophores exhibit large extinction coefficients, excellent quantum yields,  60–100 ns lifetimes, and good stability in aqueous solution (Oh et al., 2006). Pyrene also forms an excited state dimer, termed an excimer, with readily detectable emission that is red shifted by approximately 100 nm relative to the monomer. This emission wavelength switching can solve the probe background signal problem that occurs with traditional FRET molecular probes. Bis-pyrene labeled oligonucleotides have been widely used to probe DNA duplex formation and RNA folding by monitoring the monomer and excimer emission fluctuations (Lewis et al., 1997; Pairs et al., 1998;

Kostenko et al., 2001; Yamana et al., 2005; Martí et al., 2006). It is well-known that there is specific and strong coordination of mercury ions (Hg2 þ ) to the thymine-thymine (T) mismatched pairs (Xue et al., 2008), and Hg2 þ –biothiol complexes also have very high stability constants (Stricks and Kolthoff, 1953; Fuhr and Rabenstein, 1973). Based on the different affinity strength of mercury ions to T–T mismatched pairs and biothiols, some traditional fluorophores (for example fluorescein)-based molecular beacon (MB) strategies have been proposed for detection of biothiols in buffer solutions by relying on Hg2 þ -induced self-hybridization of the beacon strand (Xu and Hepel, 2011; Li et al., 2013). Therefore, based on the properties of T–Hg2 þ –T interaction and pyrene excimer, the novel T–Hg2 þ –T coordination chemistrybased pyrene excimer switching strategy should be an efficient design for time-resolved fluorescence detection of biothiols in serum. However, excimer formation is strongly distance- and geometry-dependent (i.e., it requires both close physical contact between the monomers and a close alignment of their transition dipoles (Birks, 1975)), and is therefore easily disrupted. Moreover, the formed pyrene dimer molecules are easily affected by heavy metal ions (for example Hg2 þ ) and other environmental conditions (Zheng et al., 2010; Gao et al., 2013), resulting in quenching of the pyrene excimer fluorescence emission and thus limiting their application in the sensing measurements. To address these issues, and as a continuation of our studies on cyclodextrin supermolecular inclusion-enhanced pyrene excimer emission based probe designs (Zheng et al., 2010; Gao et al., 2013), γ-cyclodextrin (γ-CD) was introduced into the sensing ensemble fabrication for biothiols assay in complicated samples. Our previous researches have shown that the formation of the host–guest complex between the pyrene dimer and γ-CD can not only offer protection to the pyrene dimer from the quenching effect of heavy metal ions and other environmental factors, improving the microenvironment conditions of the pyrene dimer to largely enhance the fluorescence intensity of the pyrene excimer, but the fluorescent lifetime of the pyrene excimer (almost 120 ns) was even longer than that of the pyrene dimer alone (Zheng et al., 2010; Gao et al., 2013). When the analyte is present, it can bind Hg2 þ and remove it from T–Hg2 þ –T complex, thereby dehybridizing the T–Hg2 þ –T structure and the pyrene excimer will be formed by coordination of the γ-CD inclusion, thus the corresponding pyrene excimer emission should be observed.

Scheme 1. Schematic description of the sensing ensemble based on cyclodextrin supramolecular inclusion-enhanced pyrene excimer switching. In the presence of thiolcontaining amino acids or peptides, they can bind Hg2 þ and remove it from T–Hg2 þ –T complex, thereby dehybridizing the T–Hg2 þ –T structure and the pyrene excimer will be formed by coordination of the γ-CD inclusion, thus the corresponding pyrene excimer emission should be observed.

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2. Experimental section

3. Results and discussion

2.1. Chemicals and Instruments

3.1. Sensing scheme

The bis-pyrene-labeled DNA probe (P1: 5′-Pyr-GAA AGA GTT TGT TGG CCC CCC TTC TTT C-Pyr-3′) was synthesized by TaKaRa Biotechnology Co., Ltd., (Dalian, China). It was dissolved in ultrapure water as stock solutions and the concentrations of oligonucleotides were accurately identified according to UV absorption at 260 nm. 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB), Reduced Lglutathione (GSH), L-cysteine (Cys), homocysteine (Hcy) and other amino acids were purchased from Sigma-Aldrich and used as received. γ-CD stock solution (100 mM) was prepared by dissolving desired amount of the materials in highly pure water. Human serum samples were supplied by the Hunan Provincial Tumor Hospital, Central South University (China). All other chemicals (AR) were commercially available and used without further purification. Solutions were prepared using deionized water (18.3 MΩ cm) produced from a Millpore Milli-Q water purification system (Billerica, MA, USA). They were deoxygenated by bubbling with purified Nitrogen. All experiments were carried out at room temperature. UV–vis absorption spectra were measured on a Hitachi U-4100 spectrophotometer (Kyoto, Japan). The steady-state fluorescence emission and the time-resolved fluorescence emission were measured on a PTI QM4 Fluorescence System equipped with the GL-3300 Nitrogen Laser (Photo Technology International, Birmingham, NJ). The pH was measured by a model 868 pH meter (Orion).

The sensing scheme of the approach is based on the T–Hg2 þ –T coordination chemistry and the traditional hairpin-structured molecular beacon (MB) with pyrene molecule labeling at its both 5′ and 3′ terminus, and at the same time γ-CD was introduced into the sensing ensemble to improve the performance of the proposed sensing strategy. As depicted in Scheme 1, the sensing ensemble is in the “off” state due to the long distance between the two labeled pyrene molecules resulted from the formation of a more stable T– Hg2 þ –T structure under the presence of Hg2 þ . In the presence of thiol-containing amino acids and peptides, Hg2 þ interacts very strongly with thiol groups and the T–Hg2 þ –T structure is dehybridized, and then the pyrene excimer will be formed due to the self-complementary 5′ and 3′ ends of the DNA probe and the cooperation interaction of γ-CD to pyrene dimer, thus resulting in largely enhancement of the pyrene excimer emission. Figs. 1 and S1 show the fluorescence spectra with GSH/Cys/Hcy and without them at room temperature (25 °C). We can see that there is a large increase in fluorescence intensity of the pyrene excimer at 480 nm for samples with GSH/Cys/Hcy, and moreover, further significant enhancement of the excimer emission can be observed after addition of γ-CD. However, the significant enhancement of the excimer emission can not be observed only in the present of γ-CD (curve d). It is worth to note that the thiol-contained amino acids (Cys, Hcy) and peptides (GSH) have no significant effect on the pyrene excimer's fluorescence emission (Fig. S2). Therefore, GSH, Cys or Hcy can be detected by using the proposed sensing ensemble at room temperature.

The mixture of P1 and Hg2 þ with a certain concentration in 10 mM Tris–HNO3 buffer solution was heated to about 85 °C for 5 min and then gradually cooled to room temperature. This mixture was allowed to be incubated at room temperature for 2 h, followed by the addition of γ-CD. 2.3. Detection of biothiols using the sensing ensemble Freshly prepared GSH/Cys stock solution or other samples was added into 400 μL of the prepared sensing ensemble solution and incubated at room temperature (25 °C) for 1 h. After reaction, the resulting solution was subjected to fluorescence measurements. For steady-state fluorescence measurement, the excitation wavelength was 345 nm and the emission wavelengths were in the range from 360 to 650 nm with both excitation and emission slits of 2.5 nm. The time-dependent fluorescence responses were recorded immediately after the addition of analysts at an excitation wavelength of 345 nm (slit 2.5 nm) and an emission wavelength of 488 nm (slit 2.5 nm). For time-resolved fluorescence emission measurement, the excitation energy was supplied by the GL-3300 Nitrogen Laser and the fluorescence responses were recorded at an emission wavelength of 488 nm. (Note: due to the high toxicity of Hg2 þ , the solutions after assays should be discarded following the waste disposal procedure.) 2.4. Real sample analysis

3.2. Optimization of the sensing ensemble One can see from Scheme 1 that three important factors should be considered in the proposed approach: γ-CD, Hg2 þ and incubation time of the sensing system. Therefore, prior to application of the probe system in the fluorescence sensing of biothiols, the effect of various factors on this system were investigated. At first, the concentration of γ-CD was optimized. As mentioned

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2.2. Preparation of the probe system

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To apply the time-resolved fluorescence measurement, biothiols in human serum was detected. The serum samples were voluntarily provided by healthy people and stored at 4 °C. Before fluorescence detection, 40 μL of the sample was mixtured with 360 μL of the sensing ensemble solution (The final concentration of P1, Hg2 þ and γ-CD is 1 μM, 5 μM and 5 mM, respectively.).

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Wavelength / nm Fig. 1. Fluorescence emission spectra of P1/Hg2 þ in the absence (curve a) and presence of GSH (curve b), and further addition of γ-CD (curve c). ‘curve d’ is the fluorescence emission spectra of P1/Hg2 þ in the presence of γ-CD. The concentrations of P1, Hg2 þ , GSH and γ-CD in 10 mM Tris–HNO3 buffer solution (pH 7.4) are 100 nM, 0.5 μM, 20 μM and 3 mM, respectively. λex ¼ 345 nm.

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above, γ-CD is one of the most important factor affecting the performance of the probe system. Other than the fact that the stability of the stem-loop hairpin structure can be modulated via variation of the concentration of γ-CD, our previous report also showed that γ-CD can protect the pyrene excimer from the influence of Hg2 þ and enhance the fluorescence emission intensity of the excimer (Zheng et al., 2010; Gao et al., 2013). This result indicates that the inclusion interaction between γ-CD and two pyrene molecules is an equilibrium, and the performance of the sensing ensemble is dependent upon the concentration of γ-CD. That is to say, a suitable concentration of γ-CD is necessary in this probe system to guarantee high performance in the sensing of biothiols. As is shown in Fig. S3A, there is a continuous significant increase of the pyrene excimer signal until the γ-CD concentration increases up to 3 mM, indicating that more and more of the pyrene dimmer/γ-CD inclusion complex was formed due to the inclusion force of γ-CD and the inclusion interaction was reached equilibrium when the concentration of γ-CD is over than 3 mM. Effect of Hg2 þ and other bivalent metal ions including Ca2 þ , Mg2 þ , Zn2 þ , Cu2 þ , Co2 þ , Ba2 þ , Ni2 þ , Cd2 þ and Pb2 þ on the sensing ensemble was then investigated. Fig. S4 presents the difference in pyrene excimer emission at 488 nm between the solutions containing Hg2 þ and other metal ions. It can be seen from the result that there has no significant difference in the excimer emission intensity between the mentioned metal ions and the blank, while the excimer emission intensity was decreased greatly when Hg2 þ was added. These results clearly indicate that the T–T mismatch-contained DNA probe exhibited high selectivity for Hg2 þ . Then, effect of Hg2 þ concentration on the formation of excimer emission-quenched sensing ensemble was also investigated. As shown in Fig. S3B, the excimer emission intensity is decreased with the increase of Hg2 þ concentration and an equilibrium statement was reached when the concentration of Hg2 þ is over than 0.5 μM under the 100 nM of P1 and 3 mM of γ-CD. In view of the whole response process and explore the effect of incubation time on the performance of the sensing system, the real-time record of the response of proposed sensing ensemble to Cys/GSH was also carried out using the 488 nm excimer emission as a function of time. As shown in Fig. S3C, upon addition of Cys/ GSH, the fluorescence intensity increases quickly up to  20 min and then remains nearly constant, indicating that the reaction is completed and the sensing ensemble reaches equilibrium. In order

A

3.3. Time-resolved fluorescence assay Measurement of analytes in complex biological fluids, which contain ubiquitous endogenous components that produce a high fluorescence background (Fu et al., 2007), makes the commonly used biosensors fail to be efficient without sample pretreatment. Fig. 2A shows the steady-state emission spectra of the 10-times diluted human serum and the probe system. One can see from Fig. 3A that the 10% serum had a high autofluorescence and dominated the fluorescence spectra from 360 to 650 nm, indicating that the approach is inefficient for biothiols assay in a complex serum environment. However, most of the background fluorescence of biological molecules and organic fluorophores has a lifetime of o10 ns, but the fluorescence emission of the pyrene dimer/γ-CD inclusion complex has a much longer lifetime on the order of hundred-seconds (Gao et al., 2013). Such a big difference allows one to temporally separate the probe fluorescence signal from the background signal using the time-resolved fluorescence technique. As shown in Fig. 2B, the fluorescence intensity of the 10% human serum was decreased rapidly with increasing delay time and was even close to zero at the gate time point of 40 ns, while the long lifetime emission intensity of pyrene excimer at 488 nm was well resolved with 70% of the original value at this gate time point. This difference in the delay time of pyrene excimer fluorescence compared with that of the serum constitutes the basis for time-resolved measurement of biothiols in a complicated serum samples. 3.4. Selectivity and sensitivity With the optimized conditions as mentioned above, we successively evaluated the capability of this pyrene excimer reporterbased time-resolved fluorescent sensing ensemble for quantitative detection of thiol-containing amino acids and peptides (Here, GSH was choosed as the model). Fig. 3A shows the time-depended changes of the fluorescence emission intensity of pyrene excimer at 488 nm for the sensing ensemble in the presence of different concentrations of GSH. The fluorescence emission intensity at the gate time point of 40 ns was significantly increased with an increase in the GSH concentration. When the concentration of GSH

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to offer an enough incubation time, we have employed 1 h for all samples in following experiments.

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Wavelength / nm Fig. 2. (A) Steady-state fluorescence emission spectra of the 100 nM of P1 (curve a) and the 10% human serum (curve b); λex ¼ 345 nm. (B) Fluorescence emission intensity decays of 10% human serum (curve a), 500 nM P1 (curve b), and 500 nM P1 þ 5 mM γ-CD (curve c); the fluorescence emission intensity was recorded at 488 nm with an excitation wavelength of 330 nm supplied by the GL-3300 Nitrogen Laser. Buffer solution: 10 mM Tris–HNO3 buffer (pH 7.4).

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B Fluorescence intensity at 488 nm

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[GSH] (µM ) Fig. 3. (A) Fluorescence emission intensity decays of P1/Hg2 þ /γ-CD sensing ensemble in the presence of GSH with different concentrations (from bottom to top: 0, 1, 5, 20, 50, 100, and 150 μM, respectively). (B) Fluorescence emission intensity of P1/Hg2 þ /γ-CD sensing ensemble at the 40 ns of time gate as a function of the GSH concentration, the magnitude of the error bars was calculated from the uncertainty given by three independent measurements. The fluorescence emission intensity was recorded at 488 nm with an excitation wavelength of 330 nm supplied by the GL-3300 Nitrogen Laser. [P1]¼ 1 μM, [Hg2 þ ] ¼5 μM, [γ-CD]¼ 5 mM. Buffer solution: 10 mM Tris–HNO3 buffer (pH 7.4).

was increased to 100 μM, the significant fluorescence change can not be observed, indicating that the sensing response reached the maximum (Fig. 3B). The detection limit that is taken to be three times the standard derivation in blank solution is 0.36 μM. To determine the selectivity of the assay, we have investigated the fluorescence response to the other essential amino acids at a concentration of 20 μM under the optimum experimental conditions. The experimental results are shown in Fig. 4. It is clear that only GSH/Cys/Hcy showed significantly higher fluorescence intensity. In contrast to significant fluorescence enhancement observed for GSH/Cys/Hcy, very little change of the fluorescence intensity was observed upon addition of other amino acids, although Hg2 þ is known to have an affinity to certain N-type ligands (Corradi et al., 1993). The binding affinity of Hg2 þ to a T–T mismatch site appears to be stronger than that to all amino acids studied except for GSH/Cys/Hcy. The binding of GSH/Cys/Hcy to Hg2 þ at a T–T mismatch site is highly selective due to the high

Table 1 Analysis results of biothiols for human serum specimen. Samples (  10 dilution)

Biothiols concentration by DTNBa (μmol/L)

Biothiols concentration by the proposed methodb (μmol/L)

1 2 3

46.94 7 3.05 53.82 7 3.44 51.767 3.21

49.79 73.58 57.62 74.61 54.38 74.59

a For the detection of biothiols by DTNB method, the proteins in serum were first removed via deposition and centrifugation process and the Cys was adopted as the standard material. b The experiment was carried out according to that described in experimental section and the used linear equation obtained from Fig. 4 is F¼ 1.89Cþ 41.47. The standard deviations were calculated by three repeated measurements. The measured value multiplied by the dilution ratio is the biothiols concentration of human serum specimen.

values of the formation constants for the resulting complexes which lead to an assay with high specificity. 3.5. Assay of biothiols in human serum samples To confirm that our approach is feasible for time-resolved qualitative biothiols assay in complex biological fluids, the proposed method was preliminarily applied in the determination of biothiols in human serum samples and the obtained results were compared with that by DTNB colorimetric method (Ellman, 1959). The comparison of results was shown in Table 1. The analytical results show that the developed pyrene excimer reporter-based time-resolved fluorescent sensing method is suitable for the applications of biothiols assay in clinical area.

4. Conclusion

Fig. 4. Selectivity of the sensing ensemble toward different amino acids (20 μM). [P1] ¼ 100 nM, [Hg2 þ ] ¼ 0.5 μM, [γ-CD]¼ 3 mM. λex ¼ 345 nm. Buffer solution: 10 mM Tris–HNO3 buffer (pH 7.4).

In conclusion, we have successfully presented a novel pyrene excimer reporter-based time-resolved fluorescent sensing ensemble for serum detection of thiol-containing amino acids and peptides. Compared with the nowadays prevailing strategy of reaction and steady-state fluorescence-based biosensor construction, the use of a pyrene excimer reporter with long fluorescence lifetime decreased

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effectively the background fluorescence, thus substantially improving the performance of sensing ensemble in the complicated biological samples. The introduction of γ-CD showed a very interesting characteristic in fluorescence sensing of thiol-containing amino acids/peptides, such as not only offering protection to the formed pyrene dimers from the quenching in fluorescence, but also increasing the fluorescence emission intensity of the pyrene excimer, thereby resulting in a higher signal-to-background ratio. Considering the physiological link between thiol-containing amino acids/peptides and a variety of diseases and disease status, this novel pyrene excimer reporter-based time-resolved fluorescent sensing ensemble is expected to hold great potential for biological applications in medical research and clinical diagnostics.

Acknowledgment This work was supported by NSFC (21205143, 21475036, 91117006), China Postdoctoral Science Foundation (2014M552130), and the Open Project of State Key Laboratory of Chemo/Biosensing and Chemometrics (2013003).

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.2015.01.004.

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