MnO2 nanosheet architecture for glutathione sensing

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Cite this: DOI: 10.1039/c5cc04905a Received 14th June 2015, Accepted 30th June 2015

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A FRET-based carbon dot–MnO2 nanosheet architecture for glutathione sensing in human whole blood samples† Yuhui Wang,a Kai Jiang,ab Jiali Zhu,ac Ling Zhanga and Hengwei Lin*a

DOI: 10.1039/c5cc04905a www.rsc.org/chemcomm

A novel FRET-based sensing platform employing fluorescent carbon dots and MnO2 nanosheets as energy donor–acceptor pairs is designed and fabricated for the first time, which demonstrates a promising application for the detection of glutathione in human whole blood samples with high sensitivity.

Fluorescence resonance energy transfer (FRET) is a mechanism describing non-radiative energy transfer from a luminescent donor to an energy acceptor at close distances (i.e. 1–10 nm). Owing to the advantages of high sensitivity and suitability for homogeneous detection, FRET has been widely utilized in numerous fields, such as immunoassays, nucleic acid hybridization and interaction of biomacromolecules.1 In a FRET system, the energy transfer efficiency is an important parameter for assay sensitivity, which greatly depends on the emission property of a donor and the absorption capability of an acceptor.2 Layered transition-metal dioxides or disulphides (LTMDs, for instance, MoS2, WS2 and MnO2) are a class of two-dimensional (2D) nanomaterials that possess fascinating characteristics, including large surface areas, and semiconducting and energy harvesting properties, and have been explored for widespread applications in energy generation,3 sensing,4 photocatalysis5 and photothermal therapy.6 Due to their excellent light absorption capability and fast electron transfer rate, LTMDs have been verified as promising fluorescence quenchers and attracted increasing attention in constructing FRET-based bio/chemical sensing platforms. For example, Liu’s group firstly developed MnO2 nanosheet-based FRET biosensors for intracellular glutathione (GSH) detection using lanthanide ion co-doped upconversion phosphors (UCPs) a

Ningbo Institute of Materials Technology & Engineering (NIMTE), Chinese Academy of Sciences, Ningbo 315201, P. R. China. E-mail: [email protected] b Department of Applied Physics, Chongqing University, Chongqing 401331, P. R. China c School of Materials Science and Engineering, Shanghai University, Shanghai 200444, P. R. China † Electronic supplementary information (ESI) available: Experimental section and relevant figures and tables. See DOI: 10.1039/c5cc04905a

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as the energy donors in 2011.7 Thereafter, Yuan et al. made a UCP-MnO2 FRET model to be used as a label-free homogeneous biosensing platform through linking different aptamers or peptides to UCPs.8 Nonetheless, relatively low quantum yields and overheating effects induced by near-infrared light limit further applications of UCPs.9 Meanwhile, layered transitionmetal disulphides (e.g. MoS2 and WS2) as energy acceptors were exploited as well, especially by the groups of Fan and Yu.10 In their systems, dye-modified ssDNAs were designed as energy donors, which self-assembled with disulphide nanosheets via p–p stacking to form nanoprobes and were applied for the detection of biomolecules.10 However, the emission of organic dyes is often unstable and enables easy photobleaching. To overcome these above-mentioned drawbacks of donors, Tang et al. recently constructed a FRET-based sensor using persistent luminescent nanoparticles (PLNPs) and MnO2 nanosheets as the energy donor–acceptor pairs.11 Owing to the elimination of background noise in excitation, the utilization of PLNPs significantly improved the signal-to-noise ratio. But, the relatively complicated preparation procedure and poor monodispersity in aqueous solution might restrict further applications of PLNPs. Therefore, it is still highly desirable to seek new fluorescent donors to develop LTMD-based FRET assays. Fluorescent carbon-dots (C-dots) are a new class of luminescent nanomaterials that have attracted broad attention in recent years.12 Compared to the conventional semiconductor quantum dots, organic fluorescent dyes and UCPs, C-dots have many advantages, such as easy preparation, no/low toxicity and high photostability.13 Thus, C-dots have demonstrated many potential applications including sensing, bioimaging, catalysis, and optoelectronic devices.12 Inspired by the above information, we suppose that C-dots and LTMDs might be superior energy donor– acceptor pairs for fabricating FRET-based homogeneous assays. To explore the usefulness of the above concept, herein, we construct a FRET-based sensing platform using C-dots and MnO2 nanosheets as the energy donor–acceptor pairs. To the best of our knowledge, this is the first example of FRET between fluorescent C-dots and LTMDs. The as-fabricated probe further

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Scheme 1 Schematic illustration of the preparation of C-dots–MnO2 nanosheets and the principle of the FRET-based sensing platform for GSH (not to real scale).

demonstrates an application for the detection of GSH, the most abundant thiolated tripeptide in mammals. Since the abnormal levels of GSH in the human body may imply many diseases, such as cancer, liver damage and caducity,14 the development of highly sensitive and selective methods for GSH detection has been a long-term task. Scheme 1 shows the procedure for fabricating the FRETbased C-dot–MnO2 nanosheet sensing platform. Firstly, a C-dot–MnO2 composite is prepared by the reduction of KMnO4 with 2-(N-morpholino)ethanesulfonic acid (MES) (i.e. producing MnO2 nanosheets) in C-dot aqueous buffer (see details in the ESI†), and meanwhile C-dots deposit on the surface of MnO2 nanosheets. The fluorescence emission of C-dots is then quenched due to FRET from C-dots to MnO2. Secondly, upon the introduction of GSH, MnO2 nanosheets are reduced to Mn2+ (i.e. decomposition of MnO2) and thus the fluorescence of C-dots recovers. To fabricate the as-described sensing platform, C-dots are firstly prepared. Given the possible chelation and electrostatic interactions between MnO2 and carboxyl groups (–COOH), and thus achieving an effective FRET,7 carboxyl functionalized C-dots were designed and prepared based on our previous method (see the ESI† for details).15 TEM observation clearly confirms the successful obtainment of C-dots, which display a narrow diameter distribution (ca. 3–5 nm) and excellent monodispersity in aqueous solution (Fig. 1A). The fluorescence emission of the C-dots shows a similar characteristic of excitation-dependence (i.e. shifts of emission maxima upon changing the excitation wavelength) to those in many previous reports (Fig. 1B).12 The UV-vis spectrum of the C-dots displays a strong absorption peak at 325 nm and a weak absorption band at ca. 370–500 nm (Fig. S1A, ESI†). The strong absorption peak at 1695 cm 1 in the FT-IR spectrum of C-dots verifies abundant carboxyl groups being modified on the surface of C-dots (Fig. S2, ESI†). It has also been confirmed that the C-dots exhibit highly stable emission in a relatively wide pH range (from 4 to 11, Fig. S3A, ESI†) and possess high photostability under continuous irradiation by ultraviolet light (Fig. S3B, ESI†). In addition, the emissive quantum yield of the C-dots was measured to be 0.2. All these results imply that the as-prepared C-dots should be superior energy donor candidates in FRET. The energy acceptor, i.e. MnO2 nanosheets, can be facilely prepared according to the literature reports.7,11 As shown in Fig. S4A (ESI†), the TEM image shows typical nanosheet

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Fig. 1 (A) TEM image of the as-prepared C-dots. (B) Fluorescence emission spectra of the C-dots at different excitation wavelengths. (C) TEM image of the C-dot–MnO2 nanosheet composite. (D) An enlarged photograph of the selected areas in (C).

morphology of MnO2. In Fig. S4B (ESI†), MnO2 nanosheets exhibit a wide absorption spectrum from ca. 250 to 600 nm, indicating a good spectral overlap with the emission of the C-dots and thus as promising energy acceptors. Furthermore, the acquisition of the C-dot–MnO2 composite is observed in TEM images, which clearly show that the C-dots are well interspersed on the surface of MnO2 nanosheets (Fig. 1C and D). Subsequently, concentrations of the donor (i.e. the C-dots) and the acceptor (i.e. MnO2) are optimized to achieve the best sensing performance. As shown in Fig. 2, fluorescence of the C-dots (10 mg mL 1) is significantly quenched with the gradual addition of KMnO4 from 0 to 0.3 mM (the same concentration of MnO2 nanosheets being produced), and then reaches a plateau even at higher concentrations. The maximum fluorescence quenching efficiency of this system is observed to be ca. 87% (Fig. 2B). Therefore, 10 mg mL 1 of the C-dots and 0.3 mM of MnO2 are taken and used to construct the FRET-based sensing platform in this study. Next, the fluorescence lifetimes of the C-dots and the C-dot–MnO2 composite are measured and calculated to be 10.4 ns and 2.1 ns, respectively (Fig. S5 and Table S1, ESI†).

Fig. 2 (A) Fluorescence responses of the C-dots (10 mg mL 1) with different concentrations of MnO2 nanosheets (lex = 325 nm). (B) Fluorescence quenching efficiency versus concentrations of MnO2 nanosheets.


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Based on the well-known equation for evaluation of FRET efficiency, i.e. E = 1 tD–A/tD,16 where E is the energy transfer efficiency, tD and tD–A are fluorescence lifetimes of the donor and the donor–acceptor pair, respectively, the energy transfer efficiency of the C-dot–MnO2 composite is calculated to be 79.8%. The good agreement of the calculated FRET efficiency (79.8%) and observed fluorescence quenching efficiency (87%, Fig. 2B) demonstrates a FRET process from C-dots to MnO2 nanosheets. As shown in the following equation (eqn (1)),7 GSH can be oxidized to glutathione disulfide (GS-SG) by MnO2, and meanwhile it is itself reduced to Mn2+. 2GSH + MnO2 + 2H+ - GS-SG + Mn2+ + 2H2O

(1)

This reaction actually lays the foundation for the analysis of GSH. Due to the disappearance of the MnO2 acceptor, the FRET is inhibited and thus results in fluorescence restoration of the C-dots. To employ this sensing platform (i.e. C-dot–MnO2 composite), a kinetic curve of GSH reacting with the composite was firstly investigated by monitoring the fluorescence changes as a function of time. As seen in Fig. S6 (ESI†), the fluorescence of this system (in the presence of 0.6 mM GSH) gradually increases with the elongation of time and remains constant after eight minutes. Thus, a ten-minute equilibrium is always considered for the subsequent GSH detection experiments. To demonstrate the application of the C-dot–MnO2 composite for the detection of GSH, various concentrations of GSH are introduced. As shown in Fig. 3A and Fig. S7 (ESI†), the fluorescence of the system enhances with an increase in GSH concentrations and the maximum is reached at 0.6 mM. This observation is easily understood based on eqn (1), i.e. two moles of GSH (i.e. 0.6 mM) being required to reduce one mole of MnO2 (i.e. 0.3 mM). More importantly, the fluorescence enhancement is found to be linearly dependent on the concentrations of GSH ranging from 0.2 to 600 mM with a perfect correlation coefficient of 0.9934 (Fig. 3B), indicating the potential of this system for quantitative analysis of GSH. Based on the obtained linear relationship (Fig. 3B), a limit of detection (LOD) for GSH is consequently calculated to be 22 nM according to the 3s (signalto-noise) criteria.17 Note that the LOD (i.e. sensitivity) of our system is found to be at least one order of magnitude lower than those of previous FRET-based probes that similarly used MnO2

Fig. 3 (A) Fluorescence restoration of the C-dot–MnO2 nanosheet sensing platform upon addition of various concentrations of GSH (lex = 325 nm). (B) Relationship and linear fitting between the fluorescence restoration of the C-dots and the concentrations of added GSH (F0: fluorescence intensity of the C-dot–MnO2 composite; F: fluorescence intensity of the C-dot– MnO2 composite in the presence of GSH).


Fig. 4 Fluorescence responses [(F F0)/F0] of the C-dot–MnO2 nanosheet sensing platform towards GSH and interferents (F0: the fluorescence intensity of the sensor; F: the intensity of the sensor in the presence of interferents).

nanosheets as energy acceptors.7,11,18 Additionally, the sensitivity of this proposed method is also found to be comparable to those of the other non-fluorescence approaches, such as colorimetry, electrochemistry, spectrophotometry and HPLC (Table S2, ESI†). The competitive LOD observed here may be attributed to the excellent quenching capability of MnO2 nanosheets to the fluorescent C-dots and the high FRET efficiency between them. In addition, the as-prepared C-dots possess highly pH and optical stability, good water-solubility and respectable fluorescence quantum yield, making them ideal candidates for sensing usage. As a superior probe, sufficient selectivity is another significant requirement. Thus, interferences of many common species, especially in the human body, including metal ions (Na+, K+, Mg2+, Ca2+, Zn2+ and Fe3+), amino acids (alanine, glutamine, histidine and glutamic acid), glucose, vitamin C (vC), cysteine (Cys), and homocysteine (Hcy) are widely investigated (their corresponding concentrations are listed in Table S3, ESI†). As shown in Fig. 4, the as-developed sensor exhibits great fluorescence enhancement in the presence of GSH (0.6 mM), while no or only slight fluorescence changes are caused by all the investigated interferents. It should point out that high concentrations of the small molecular thiols (i.e. Cys and Hcy) and reducing agents (i.e. vC) could also induce certain fluorescence recovery. But, considering the concentrations of these interferents in human whole blood being generally much lower than that of GSH (Table S3, ESI†),19 the C-dot–MnO2 composite actually represents a highly selective probe towards GSH in human whole blood samples. Finally, the practicability of the as-developed sensing platform for GSH in human whole blood samples is examined. To achieve a more reliable result, 100-fold diluted human plasma was firstly taken as the assay matrix to build a working curve. As shown in Fig. S8A (ESI†), similar fluorescence recoveries with the addition of GSH to that in buffer are observed. And an excellent linear relationship between the enhancement of fluorescence and the concentrations of GSH is found (Fig. S8B, ESI†). These results indicate high robustness of the as-developed method for GSH, even in a complex biological matrix. Then, the sensor was used for the detection of GSH in human whole blood samples.

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After pretreating the whole blood using a routine procedure (details in the ESI†),19a the 100-fold dilution samples were directly added into the sensing system and the corresponding fluorescence intensities were recorded. The concentrations of GSH can be readily obtained based on the working curve in the plasma matrix (three samples were tested and the results are summarized in Table S4, ESI†). Taking the dilution into account, the GSH concentrations of the three human whole blood samples are determined to be 810, 900 and 840 mM, respectively, all being consistent with the reported results by HPLC.19a Moreover, standard addition experiments are performed as well, and 88.5% to 101.9% recoveries are observed with acceptable relative standard deviations (Table S4, ESI†), further indicating the reliability of this developed sensing system. In summary, a novel FRET-based sensing platform using fluorescent C-dots and MnO2 nanosheets as the energy donor– acceptor pairs is designed and fabricated for the first time. This sensing system demonstrates a practical application for the highly selective and sensitive detection of GSH in human whole blood samples. This study reveals that C-dots–MnO2 nanosheets shall be superior energy donor–acceptor pairs for fabricating FRET-based assays. More interestingly, this work might open up new possibilities in developing other LTMD-based bio/chemical sensing platforms through linking various functional ligands (e.g. peptides or aptamers) onto C-dots. This work was financially supported by the National Natural Science Foundation of China (21305152 and 21277419), the Zhejiang Provincial Natural Science Foundation (LR13B050001), and the Ningbo Science and Technology Bureau (2014B82010).

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