Biosensors and Bioelectronics 65 (2015) 83–90

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Onsite naked eye determination of cysteine and homocysteine using quencher displacement-induced fluorescence recovery of the dualemission hybrid probes with desired intensity ratio Kan Wang 1, Jing Qian 1, Ding Jiang, Zhengting Yang, Xiaojiao Du, Kun Wang n Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 24 July 2014 Received in revised form 29 September 2014 Accepted 30 September 2014 Available online 5 October 2014

Simple, inexpensive, portable sensing strategies for those clinically relevant molecules have attained a significant positive impact on the health care system. Herein, we have prepared a dual-emission ratiometric fluorescence probe with desired intensity ratio and demonstrated its efficiency for onsite naked eye determination of cysteine (Cys) and homocysteine (Hcy). The hybrid probe has been designed by hybridizing two differently sized CdTe quantum dots (QDs), in which the red-emitting CdTe QDs (rQDs) entrapped in the silica sphere acting as the reference signal, and the green-emitting CdTe QDs (gQDs) covalently attached on the silica surface serving as the response signal. When 1,10-phenanthroline with strong coordination ability to Cd atoms in gQDs was introduced, the fluorescence of the gQDs was effectively quenched, while the fluorescence of the rQDs stayed constant. Upon exposure to different contents of Cys or Hcy, the fluorescence of gQDs can be recovered gradually due to the displacement of the quencher. Based on the background signal of rQDs, the variations of the sensing system display continuous fluorescence color changes from red to green, which can be easily observed by the naked eye. The assay requires ∼20 min and has a detection limit of 2.5 and 1.7 μM for Cys and Hcy, respectively. Furthermore, we demonstrate that this sensing scheme can be fully integrated in a filter paper-based assay, thus enabling a potential point-of-care application featuring easy operation, low power consumption, and low fabrication costs. & 2015 Elsevier B.V. All rights reserved.

Keywords: Dual-emission hybrid probes Ratiometric fluorescence Naked eye determination Cysteine Homocysteine

1. Introduction Ratiometric fluorescent sensors, which allow the measurement of changes in the ratio of the fluorescence intensities at two wellresolved wavelengths, have received intense attention in recent years (Wu et al., 2012; Chen et al., 2013; Tyrakowski and Snee, 2014). In comparison with single-wavelength measurements, ratiometric fluorescence technique can provide built-in correction for environmental effects (Zhuang et al., 2014; Diaz et al., 2012; Lv et al., 2011), eliminates the fluctuation of excitation light intensity and the probe concentration (Dubertret et al., 2002; Deniz et al., 2001; Li et al., 2011), and thus possesses advantages in terms of improved sensitivity and accuracy. Till now, most reported ratiometric sensors using fluorescent organic dyes have been constructed based on the internal charge transfer (Das et al., 2012), native chemical ligation reaction (Lv et al., 2014), and fluorescence n

Corresponding author. Fax: þ 86 511 88791708. E-mail address: [email protected] (K. Wang). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.bios.2014.09.093 0956-5663/& 2015 Elsevier B.V. All rights reserved.

resonance energy transfer (FRET) (Long et al., 2011). However, there is difficult in choice of dyes, since many of them remain associated with some disadvantages including low fluorescence quantum yield, weak resistance to photo-bleaching, narrow excitation and broad emission bands, and especially the complexity of organic molecular synthesis and purification (Wu and Chiu, 2013; Zong et al., 2011). Therefore, there remains a great challenge to construct simple, low-cost, and high-efficient ratiometric fluorescence probes for practical use. Fluorescent quantum dots (QDs) have been demonstrated to exhibit excellent optical properties, such as broad excitation spectra, high fluorescence quantum yield, and strong resistance to photo bleaching which are better suited as fluorescence probe than traditional organic dyes (Liu et al., 2014; Reiss et al., 2009; Wu et al., 2014). More importantly, size-dependent emission of QDs affords the convenience of incorporating differently sized QDs into a complex material to create a multicolor system, which is central to the ratiometric fluorescence detection (Gui et al., 2013; Yao et al., 2013). Generally, dual-emission is observed in core– satellite hybrid sphere containing two different species, in which one species is entrapped into the nanosphere serving as the

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internal standard, and the other is loaded on the surface of the nanosphere acting as the response signal. Following the “turn off” detection mode, several QDs-based dual-emission fluorescence probes with suitable intensity ratios have been demonstrated to be promising for environmental analysis, e.g., the visual detection of Cu2 þ (Zhu et al., 2012; Wang et al., 2014) and Hg2 þ (Sun et al., 2012), and the homeland security, e.g., the visual detection of trinitrotoluene (Zhang et al., 2011). In principle, such simple, inexpensive, portable sensing strategies are more suitable and affordable for point-of-care (POC) testing, especially in resourcelimited settings such as developing countries and remote communities (Zhu et al., 2014; Yang et al., 2012; Mu et al., 2014). Displacement assay approach is the most popular way to detect analytes through “turn on” fluorescence signaling. It is reported that displacement directed chemosensing of analytes in fact improves the selectivity by many folds (Wang et al., 2012). By utilizing the dual-emission organic dyes, recent reports mainly focus on the displacement of quencher leading to the construction of efficient anion sensor (Kaur et al., 2013; Kumar et al., 2013; Singh et al., 2014). Due to the aforementioned disadvantages of the organic dyes, design and synthesis of the QDs hybrid probes and search for novel “turn on” detection principles, to perform the onsite visual detection of those clinically relevant molecules for POC diagnosis and disease screening, is of great significance. Thiol-containing amino acids (aminothiols), such as cysteine (Cys) and homocysteine (Hcy) which are structurally similar and metabolically linked, serve vital functions in human tissues including protein synthesis, detoxification, and metabolism (Chen et al., 2010). However, abnormal levels of aminothiols are relative to many human diseases. For example, a deficiency of Cys associated with hematopoiesis decrease, muscle and fat loss, psoriasis, slow growth in children, liver damage, skin lesions, hair depigmentation, edema, and so forth (Shahrokhian, 2001). The elevated Hcy level in blood is known to be directly linked to several disorders including cardiovascular and Alzheimer's diseases, neural tube defects, and osteoporosis (Klee, 2000; Seshadri et al., 2002; Refsum et al., 2004). Accordingly, the sensitive determination of these aminothiols in plasma and urine samples is of considerable importance and significant interest due to its promising application for the early disease screening and diagnosis (Yin et al., 2013; Jung et al., 2013). Classical determination of these aminothiols is generally accomplished by the high-performance liquid chromatography (HPLC) (Nolin et al., 2007), capillary electrophoresis (CE) (Kubalczyk et al., 2014), mass spectrometry identification (MS) (Vellasco et al., 2002), and electrochemistry (CE) (Wei et al., 2011). Of various sensing protocols, the color change observed by the naked eye is considered to be the most inexpensive and convenient way to determine those clinically relevant molecules for diagnosis onsite. To date, design of such sensors for Cys or Hcy still represents a great challenge. Herein, we demonstrate a new concept for the onsite naked eye deterimination of Cys and Hcy. A dual-emission fluorescent hybrid sphere by using two differently sized CdTe QDs was prepared with desired intensity; the red-emitting CdTe QDs (rQDs) were entrapped in the silica while the green-emitting CdTe QDs (gQDs) were attached on the silica surface. Upon the introduction of 1,10phenanthroline (Phen) with strong coordination ability to Cd atoms in gQDs, the green fluorescence of the attached gQDs was effectively quenched, while the red fluorescence of the entrapped rQDs stayed constant. In the presence of different contents of Cys or Hcy, however, the fluorescence of gQDs is recovered gradually because of the strong binding preference of gQDs for analytes, thus, exhibited continuous fluorescence color changes induced by Cys and Hcy without the need of elaborate equipment.

2. Experimental 2.1. Reagents Tetraethylorthosilicate (TEOS), 3-aminopropyltriethoxysilane (APTS), 3-mercaptopropionic acid (MPA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and amino acids were purchased from Sigma-Aldrich. Tellurium powder (99%), CdCl2  2.5H2O, ammonium hydroxide (NH3  H2O, 25%), NaBH4 (99%), tris(hydroxymethyl)methyl aminomethane, ethanol, and Phen were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Two differently sized CdTe QDs (gQDs and rQDs) capped with MPA containing carboxylic group were obtained according to a previous report (Zhang et al., 2010). All the reagents were used as purchased without further purification. Double-distilled water was used throughout the study. 2.2. Apparatus The transmission electron microscopy (TEM) images were taken with a JEOL 2100 TEM (JEOL, Japan) at 200 kV. UV–vis absorption spectra were measured by UV-2450 spectrophotometer (Shimadzu, Japan). Fluorescence spectra were recorded on a Hitachi F-4500 fluorescence spectrophotometer (Tokyo, Japan). All the photographs were taken using a Canon digital camera (IXUS 230 HS, China) under the illumination of a 365 nm UV lamp. 2.3. Preparation of amino-functionalized rQDs@SiO2 spheres The rQDs were loaded inside silica spheres by a modified Stöber method (Zhang et al., 2011). Briefly, 0.5 mL of rQDs solution and 40 mL of ethanol were mixed in a 100 mL flask and stirred for 10 min, 100 μL of APTS was then added to the above solution and stirred for 6 h. Then, 0.5 mL of TEOS and 0.5 mL of NH3  H2O was introduced, and the mixture was stirred for another 12 h. Finally, 100 μL of APTS was added under vigorous stirring and reacted for 12 h in order to modify the silica surface with amino groups. The as-prepared amino-functionalized rQDs@SiO2 was subjected to several cycles of precipitation by centrifugation and washed with ethanol and water to remove the unreacted chemicals. 2.4. Preparation of dual-emission rQDs@SiO2@gQDs Hybrid spheres In a typical synthesis, 6 mL of EDC/NHS (2 mg mL  1 for each), 12 mL of H2O and 2.4 mL of gQDs solution were added in a 50 mL flask. After stirring for 15 min at room temperature, the asobtained amino-functionalized rQDs@SiO2 spheres were added into the mixture which was stirred vigorously for 4 h in the dark. Finally, the rQDs@SiO2@gQDs hybrid spheres with dual-emission fluorescence were collected by centrifugation and washing and redispersed in 10 mL of water for later use. 2.5. Procedures of fluorescence spectrometric and visual detection 50 μL of the as-prepared rQDs@SiO2@gQDs hybrid probes, 90 μL of 1 mM Phen, a certain volume of Cys or Hcy were added into a 0.5 mL tube, the mixture was diluted with Tris–HCl buffer (pH 7.4, 50 mM) to a final volume of 200 μL and reacted for 20 min at room temperature to obtain solution A. To perform the visual fluorescence detections, solution A was transferred to a series of miniwells and the photographs were obtained using a digital camera under UV illumination (λex ¼365 nm) in a dark box. To perform the spectrometric analysis, solution A was diluted to 3 mL with Tris–HCl buffer (pH 7.4, 50 mM) and the fluorescence spectra

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of the diluted mixture solutions were recorded using a fluorescence spectrophotometer (λex ¼365 nm).

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545 nm versus that of rQDs (Ir) at 645 nm remained no significant change in 2 h under an excitation at 365 nm (Fig. S2). That is, these hybrid spheres can exhibit good photostability, making them be applied in the following Cys and Hcy detection.

3. Results and discussion 3.2. Working principle for Cys and Hcy determination 3.1. Characterization of the core–satellite hybrid spheres

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The gQDs (curve a) and rQDs (curve b) had a maximum absorption peak at 497 and 598 nm (Fig. S1), and emitted strong fluorescence with the maximal emission wavelength at 545 and 645 nm, respectively (Fig. 1A). According to the reported equation (Yu et al., 2003), the diameter of the gQDs and rQDs was calculated to be 2.40 and 3.7 nm, respectively. Photographs of the aqueous dispersed gQDs (a) and rQDs (b) under UV illumination showed they exhibited an intense green and red fluorescence, respectively (inset of Fig. 1A). The rQDs@SiO2 was first prepared by a modified Stöber method; afterwards, APTS was coupled to the hydroxyl groups on silica surface to produce an amino-terminated self-assembled monolayer. Subsequently, the carboxylic groups on MPA-capped gQDs were activated with EDC/NHS, and the activated gQDs were covalently linked to the surface of rQDs@SiO2 through the reaction between amino and carboxyl to form the core-satellite rQDs@SiO2@gQDs hybrid spheres. The TEM images (Fig. 1B) show that the as-prepared rQDs@SiO2 spheres had a chemically clean and homogenized structure with a diameter of ∼150 nm. When excited using a UV lamp, the aqueous rQDs@SiO2 exhibited a strong red emission, similar to that of the original rQDs. The presence of the silica shell has two unique benefits. On one hand, it can prevent the direct contact of the rQDs with the external solvents during the linking period and the subsequent detection, hence providing a stable and reliable reference signal; on the other hand, it can avoid the FRET process between the reference rQDs and the response gQDs, thus enhancing the optical and structure stability of the hybrid probe. The rQDs@SiO2 spheres were further amino-functionalized so that the MPA-capped gQDs with carboxylic groups could be covalently linked on the surface through a condensation react. After the covalent linking process, a crude surface was observed for rQDs@SiO2@gQDs (Fig. 1C), suggesting that the gQDs have been successfully linked on the silica shells. The obtained hybrid spheres had dual unique, well-resolved emission peaks at 545 nm and 645 nm, which were ascribed to the respective emission of gQDs and rQDs (curve c in Fig. 1A). When excited under UV lamp, the as-prepared rQDs@SiO2@gQDs hybrid spheres solution (photograph c inset in Fig. 1A) showed a strong yellow–green emission, differing from that of the original gQDs and rQDs. In addition, the fluorescence intensity of gQDs (Ig) at

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The response of the ratiometric fluorescence probe to Cys and Hcy has been monitored to prove the working principle, as shown in Fig. 2. The hybrid probe exhibited well-resolved dual emission bands under a single wavelength excitation (Fig. 2A). When 30 μM of Phen was introduced (Fig. 2B), the fluorescence of the gQDs which were attached on the surface of the silica spheres was quenched completely. The affinity of the two nitrogen atoms of Phen with Cd atoms made Phen attached onto the gQDs surface by replacing the original MPA ligands. The significant quenching of the gQDs fluorescence in the presence of Phen was mainly caused by the static quenching and the formation of the non-fluorescent ground-state complex (Chen et al., 2013; Hu et al., 2014). Interestingly, when Cys (Fig. 2C) or Hcy (D) was added into the hybrid spheres–Phen system, there was ∼37 fold and ∼49 fold enhancement in the fluorescence intensity at 545 nm compared to that of the hybrid spheres–Phen system, respectively. Cys and Hcy were able to displace Phen because the potent binding capability of Cys and Hcy (–SH, –NH2 and –COOH goups) to gQDs was much stronger than that of Phen (Chen et al., 2013). The recovered fluorescence resulted from the analytes-induced displacement of the quencher and the formation of the stable and fluorescent Cyscapped gQDs (Fig. S3) and Hcy-capped gQDs (data not shown) was a possible way for the fluorescence recovery (Chen et al., 2013). During the fluorescence quenching and recovering of the gQDs upon the exposure of Phen and analytes, the fluorescence of rQDs always stayed constant. That is, the rQDs encapsulated in the silica core could be served as the reference signal, thus provided a builtin correction for environmental effects, while the gQDs covalently linked on the silica surface could be served as the response signal for Cys and Hcy sensing. Upon the “on-off-on” state of the green fluorescence, a ratiometric fluorescence response is therefore realized for Cys or Hcy detection (illustrated as inset of Fig. 2). The changes in the intensity ratio of the two emission wavelengths resulted in a noticeable fluorescence color change (photographs inset in Fig. 2A–D), and thus facilitating the visual detection of Cys and Hcy onsite. The ratio of the emission peaks could be tuned by changing the volume ratio of gQDs:rQDs (Fig. S4), which would help to design the ratiometric fluorescence probe with high color difference acuity for the naked eye identification. Control experiments in this study showed that the volume ratio of gQDs:rQDs ¼4.8:1

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Fig. 1. (A) Fluorescence spectra of gQDs (a), rQDs (b), and rQDs@SiO2@gQDs hybrid spheres (c). Inset: The corresponding photographs under UV lamp (λex ¼ 365 nm). (B) TEM images of rQDs@SiO2 at low and high magnification (inset). (C) TEM image of rQDs@SiO2@gQDs hybrid spheres. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

K. Wang et al. / Biosensors and Bioelectronics 65 (2015) 83–90

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Fig. 2. Fluorescence spectrum of the hybrid spheres (A), hybrid spheres–Phen (30 μM) (B), hybrid spheres–Phen (30 μM)–Cys (1000 μM) (C), hybrid spheres–Phen (30 μM)– Hcy (800 μM) (D). Inset: Schematic illustration of the working principle for the visual fluorescence detection of Cys/Hcy.

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Fig. 3. (A) Fluorescence spectra of the hybrid spheres upon exposure to different concentrations of Phen (from a to j: 0, 1, 2, 5, 10, 15, 20, 25, 30, and 35 μM). (B) The effects of pH on the fluorescence quenching efficiency of the probe at 545 nm. (C) The effects of pH on the recovered fluorescence intensity of the hybrid spheres–Phen system. Inset: Plots of the corresponding recovered fluorescence intensity at 545 nm. (D) The effects of time on the recovered fluorescence intensity of the hybrid spheres–Phen (from a to g: 0, 1, 5, 10, 15, 20, and 30 min). Inset: Plots of the corresponding recovered fluorescence intensity at 545 nm.

K. Wang et al. / Biosensors and Bioelectronics 65 (2015) 83–90

would produce the hybrid probe displaying distinguishable colors to a small increase of Cys and Hcy concentration. Therefore, the hybrid sphere with this desired intensity ratio was used as the ratiometric fluorescence probe for further analysis. 3.3. The optimization of important factors

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quenching efficiency of the nanoprobes at 545 nm is 98.59%, 98.72%, 98.68%, 95.16%, 94.67%, and 97.85%, for the solution with a pH value of 6.5, 7.0, 7.4, 8.0, 8.5, 9.0, accordingly. Although the effect of pH on the quenching efficiency of gQDs by Phen was not obvious, it was quite evident on the recovered fluorescence intensity of gQDs. As revealed from Fig. 3C, with the increasing value of the solution pH, the recovered fluorescence intensity increased gradually and reached its maximum at pH 7.4. Beyond this value, the recovered fluorescence intensity began to fall down significantly. Thus, the Tris–HCl buffer with a pH value of 7.4 was chosen to be the optimized condition for the following study. The formation of probe–Phen complexes was very fast and the reaction was accomplished within a couple of seconds, whereas the fluorescence recovery of gQDs was relatively slow and thus needed to be investigated. As shown as Fig. 3D, the introduction of Cys gradually displaced Phen to form the stable luminescence gQDs and the fluorescence intensity at 545 nm was steadily increasing over time. The maximal fluorescence recovered signal was achieved at 20 min and then kept stable beyond this time. These results indicated that 20 min was appropriate for the reaction between Cys and hybrid spheres–Phen system.

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In order to receive a high sensitivity for the subsquent detection, the effects of the Phen concentrations on the quenching efficiency of gQDs were studied. Fig. 3A exhibits the fluorescence spectra of the nanoprobe in different concentrations of Phen from 0 to 35 μM in 50 mM Tris–HCl buffer (pH 7.4). Obviously, the fluorescence of the embedded rQDs was stable, while the fluorescence intensity of gQDs reduced gradually with the increasing concentrations of Phen. When the concentration of Phen was increased to 30 μM, the fluorescence of the gQDs was totally quenched. Therefore, the concentration of Phen was chosen to be 30 μM for further experiments. The fluorescence quenching efficiency of the hybrid spheres– Phen system was also monitored in 50 mM Tris–HCl solution with different pH values. As displayed in Fig. 3B, the corresponding

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Fig. 4. Fluorescence spectra and the corresponding photographs of the hybrid spheres–Phen system (A) and gQDs–Phen system (B) upon the exposure to different concentrations of Cys (from a to n: 0, 5, 10, 20, 30, 40, 60, 80, 100, 200, 300, 500, 800, and 1000 μM). Inset of A: Linear relationship for Cys detection (n¼ 3). (C) Fluorescence spectra and the corresponding photographs of the hybrid spheres–Phen system upon the exposure to different concentrations of Hcy (from a to m: 0, 5, 10, 20, 30, 40, 60, 80, 100, 200, 300, 500, and 800 μM). (D) Linear relationship for Hcy detection (n¼ 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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3.4. Naked eye determination of Cys and Hcy On the basis of the hybrid spheres–Phen system with Cys and Hcy as the fluorescence signal enhancer, the dose response of the sensing system to Cys and Hcy has been examined in detail. Upon the addition of Cys with different concentrations (Fig. 4A), the intensity of the emission from the gQDs was increased successively with the increasing concentration of Cys, whereas the fluorescence intensity of rQDs stayed constant. The fluorescence intensity of gQDs ceased to increase beyond 1000 μM of Cys, indicating the reaction was nearly saturated at this concentration. By analyzing the ratio of the fluorescence intensity with the concentrations of Cys, we obtained a linear relationship (inset in Fig. 4A) between the (Ig/Ir)/(Ig/Ir)0 value and the logarithm of the concentration of Cys (cCys) over a range of 20– 1000 μM, where (Ig/Ir)0 is the relative fluorescence intensity of the hybrid spheres–Phen system without Cys. The linear curve fitted a regression equation of (Ig/Ir)/(Ig/Ir)0 ¼  19.53þ19.59 log(cCys/μM) (R2 ¼0.9830) with a detection limit of 2.5 μM at S/N¼ 3. Owing to the changes on the relative fluorescence intensity (Ig/Ir) of the dual emission, a series of noticeable color changes could be visualized from the hybrid spheres–Phen–Cys solution (bottom of Fig. 4A). Clearly, even a slight increase of the green emission intensity resulted in an obviously distinguishable color evolution in a broad concentration range of Cys (20–1000 μM), a simple and low-cost fluorescence on-off-on assay for Cys detection can be thus realized with the naked eye. To demonstrate the advantages of the visual detection of Cys based on the ratiometric probe, the single fluorescence recovering experiments which are employed in the current fluorometry for Cys detection was also performed. With the pure gQDs as probe, the fluorescence images of the solution with a narrow Cys concentration range of 20– 80 μM can be distinguished by the naked eye, while the color of the other pure gQDs–Phen–Cys solution changed too little to tell the difference (bottom of Fig. 4B). The results indicated that the ratiometric fluorescence probe was more sensitive and reliable for visual detection of Cys in a wide concentration range, compared with that of a single fluorescence probe, although the intensity of the green emission decreased at the same level (Fig. 4B). Fig. 4C displays the fluorescence spectra and plots of the corresponding relative fluorescence intensity of the hybrid spheres–Phen system upon the exposure to different concentrations of Hcy from 0 to 800 μM. Similarly, Fig. 4D exhibits a linear dependence on (Ig/Ir)/(Ig/Ir)0 and Hcy concentration ranging from 20 to 800 μM. The calibration curve can be expressed as (Ig/Ir)/ (Ig/Ir)0 ¼  28.73 þ27.18 log(cHcy/μM) with R2 ¼0.9949, the detection limit for Hcy was calculated to be 1.7 μM (S/N ¼3). Characteristics of the present sensor along with others reported in the literatures are all summarized in Table S1. As can be seen, the

present sensor possessed a broad linear range and comparable detection limit, its sensitivity was equal or even lower than that observed in most other existed sensors based on the method using HPLC (Nolin et al., 2007), CE (Kubalczyk et al., 2014), MS (Vellasco et al., 2002), and CE (Wei et al., 2011). However, its sensitivity was approximately ∼10 fold lower than that of the ratiometric fluorescent sensors using traditional organic dyes down to 100 nM (Das et al., 2012; Lv et al., 2014). The recovered fluorescence and the color change of the hybrid spheres–Phen system offers a novel method to detect Cys and Hcy quantitatively and qualitatively. 3.5. Detection of Cys and Hcy with the hybrid spheres-modified paper Paper-based biosensor is an excellent example developed as a result of the synergy between nanotechnology and sensing scheme (Parolo and Merkoci, 2013). This kind of assay is inexpensive, easy-to-use, mainly read with the naked eye, and quite stable to a wide range of temperature and time, which seems suitable and affordable for POC diagnosis to resource-limited settings (Yetisen et al., 2013; Hu et al., 2014). To adapt this assay to paper format (Fig. 5), the hybrid spheres were immobilized onto a commonly used filter paper by a simple adsorption process (Step 1). After successively drying at room temperature, Phen was further introduced onto the hybrid spheres-modified paper (Step 2). This pretreated paper developed a red color instantaneously after applying UV illumination (Step 3). The appearance of the red color was due to the quenching of the gQDs on the silica surface as described above. The dropping of 1000 μM of Cys or 800 μM of Hcy solution on the indicating paper could make the fluorescence of the probe recover which clearly revealed a different color against the red background under the UV illumination (Step 4). Moreover, the distinct color difference of the hybrid spheres–Phen system followed by the lifted concentration of Cys or Hcy on the paper can be noted by the independent observers. On the basis of above observation, we can conclude that the indicating paper is succeeding in building the visual detection to Cys and Hcy by using the very cheap filter paper and the very simple modified procedure. 3.6. Selectivity and reproducibility of the hybrid spheres–Phen system The high selectivity is crucial for probe to be applied in the real sample detection. The potential interfering substances including amino acids, thiol-containing compounds, common ions and some molecules are investigated to evaluate the selectivity of the hybrid spheres–Phen system (Fig. S5). Obviously, the fluorescence

Fig. 5. Performing visual detection of Cys and Hcy on the surfaces using the hybrid spheres immobilized on a piece of filter paper. Bottom panel: The corresponding photographs of the sensing system with 0 (a) 50 (b), 200 (c), and 500 (d) mM of Cys and 800 mM of Hcy (e).

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recovery intensity at 545 nm was the highest for Cys and Hcy, while only a small fluorescence recovery was observed for glutathione (GSH), a main interfering substance in most Cys and Hcy sensors, and other competitive substances have barely any fluorescence change. The recovered fluorescence only resulted from the analytes-induced displacement of the quencher due to the binding capability difference. The fluorescence recovered capability followed the order of Hcy 4Cys4 4GSH may be ascribed to the steric hindrance effects and chemical coordination capability; Other thiol-containing compounds or the competitive substances with single –SH or weak amine or acid chelating sites cannot recover the fluorescent signal, because of the much less steric hindrance effects and weak binding capability (Liu et al., 2010). On account of the very weak interference of the hybrid spheres–Phen system, this strategy can be employed for the selective visual determination of Cys and Hcy. The reproducibility of the sensor was also investigated at the Cys concentration of 500 μM, and the relative standard deviation (RSD) for five independent measurements was 3.1%, indicating acceptable precision and fabrication reproducibility. As the concentrations of GSH and Hcy in human serum samples are approximately ∼20 fold smaller than the concentration of Cys (the reported concentrations of GSH, Hcy, and Cys in human serum are 7–21, 6–12, and 165.1–335.3 μM, respectively (Yang et al., 2014; Das et al., 2012)), the designed sensing system can be applied to determine Cys in real serum samples.

3.7. Detection of Cys in human serum samples To confirm the feasibility, the proposed sensor is applied to determine Cys concentrations in human serum samples. Prior to determination, an appropriate dilution (5 fold) is used in serum samples with 50 mM Tris–HCl (pH 7.4). The results obtained for the diluted serum samples are given in Table S2. The recovery test is also determined after the addition of different known amounts of Cys into the diluted serum samples. The recoveries of spiked Cys ranged from 88.0% to 104.8% with a satisfying analytical precision (RSD r5.5%), illustrating the validity of the proposed sensor, which can satisfy the requirements for the detection of Cys in human serum.

4. Conclusions A dual-emission ratiometric fluorescent probe has been designed by hybridization of two differently sized CdTe QDs with desired intensity. The entrapped rQDs act as the internal standard while the attached gQDs serve as the response probe, whose fluorescence can be significantly quenched by Phen and recovered gradually by the introduce of different contents of Cys and Hcy. Based on the red background emitted by the internal standard rQDs, one can determine Cys and Hcy in a wide concentration range by the naked-eye with high resolution and accuracy. The detection limits of Cys and Hcy were 2.5 and 1.7 μM, respectively. The fluorescence recovered capabilities of Cys and Hcy were strikingly larger than GSH and the other amino acids, making this protocol a selective assay for the detection of Cys and Hcy over GSH and other substances. Its facility has also demonstrated by constructing a more conceivable paper assay. We thus expect that the proposal could serve as a sensing basis particularly for the resource-limited settings.

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Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 21175061, 21375050, and 21405063), the Natural Science Foundation of Jiangsu province (No. BK20130481), Jiangsu Planned Projects for Postdoctoral Research Funds (No. 1301141C), Research Foundation of Jiangsu University (No. 12JDG087), and Key Laboratory of Modern Agriculture Equipment and Technology (No. NZ201109).

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.2014.09.093.

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Onsite naked eye determination of cysteine and homocysteine using quencher displacement-induced fluorescence recovery of the dual-emission hybrid probes with desired intensity ratio.

Simple, inexpensive, portable sensing strategies for those clinically relevant molecules have attained a significant positive impact on the health car...
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