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Fluorescent glutathione probe based on MnO2-phenol formaldehyde resin nanocomposite
Biosensors and Bioelectronics 77 (2016) 299–305
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Fluorescent glutathione probe based on MnO2-phenol formaldehyde resin nanocomposite Xudong Wang a, Dan Wang a, Yali Guo a, Chengduan Yang a, Xiaoyu Liu a, Anam Iqbal a, Weisheng Liu a, Wenwu Qin a,n, Dan Yan b, Huichen Guo b,n a Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province and State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, PR China b State Key Laboratory of Veterinary Etiological Biology and Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 1, Lanzhou, Gansu 730046, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 17 July 2015 Received in revised form 10 September 2015 Accepted 19 September 2015 Available online 25 September 2015
MnO2-phenol formaldehyde resin (MnO2-PFR) nanocomposite is successfully prepared by a simple chemical reduction process. The resultant MnO2-PFR nanocomposite is well characterized. The absorption band of non-fluorescent MnO2 nanosheets overlaps well with the fluorescence emission of PFR nanoparticles. The green fluorescence of PFR in this nanocomposite can be effectively quenched by fluorescence resonance energy transfer from PFR to MnO2. In the presence of glutathione (GSH), the fluorescence of PFR could be recovered due to MnO2 was reduced to Mn2 þ by GSH. The nanocomposite can be use for detecting glutathione in blood serum. & 2015 Elsevier B.V. All rights reserved.
Keywords: MnO2 Phenol formaldehyde resin Glutathione
1. Introduction The low-molecular weight thiol glutathione (γ-L-glutamyl-Lcysteinyl-glycine) (GSH) play a central role in maintaining the appropriate oxidation–reduction state of proteins, cells and organisms (Hirrlinger and Dringen, 2010). The oxidative stress has been found in many diseases (including kwashiorkor, seizure, Alzheimers’s disease, Parkinson’s disease, liver disease, cystic fibrosis, sickle cell anemia, HIV, AIDS, cancer, heart attack, stroke and diabetes) and accelerates the aging process (Wu et al., 2004). Thus, it is of great importance to design and develop the analytical strategies for the analysis of biomarkers, of oxidative stress such as GSH. During the past decades, the measurement of GSH is carried out using various detection techniques, including fluorescent spectroscopy, (Kim et al., 2011) electrochemical pulse voltammetric methods, (Safavi et al., 2009) high-performance liquid chromatography (HPLC), (Townsend et al., 2003) and colorimetric assays (Uehara et al., 2010). Among the various analytical methods, the fluorescent spectroscopy offers significant advantages over other techniques due to its generally non-destructive character, its high sensitivity and simplicity. Currently, various fluorescent n
Corresponding authors. Fax: þ 86 931 8912582. E-mail addresses: [email protected] (W. Qin), [email protected] (H. Guo). http://dx.doi.org/10.1016/j.bios.2015.09.044 0956-5663/& 2015 Elsevier B.V. All rights reserved.
probes, using organic fluorophores, (Kim et al., 2011) gold nanoclusters, (Tian et al., 2012) quantum dots (QDs), (Banerjee et al., 2009; Liu et al., 2010; Zhang et al., 2009) carbon nanomaterials, (Cai et al., 2015; Shi et al., 2014; Wang et al., 2015; Zhang et al., 2014; Zhou et al., 2012) and upconversion nanoparticles (UCPs), (Deng et al., 2011) have been developed for GSH sensing. Recently, Santra et al., designed and synthesized a simple quantum-dotbased OFF–ON fluorescent probe for detecting glutathione with high selectivity and sensitivity (Banerjee et al., 2009). However, the toxicity of these QDs has to be remedied if one wants to design fluorescent GSH probes use inside the biological cells due to QDs contain Cd et al heavy metal ions. A sensitive turn-on sensor for GSH in the cell samples based on the recovered fluorescence of the gold nanoclusters Hg2 þ system was reported by Zhu and coworkers (Tian et al., 2012). However, Hg is a kind of wellknown toxicity and potential environmental hazard of the heavy metals. Recently, Yu’s group has reported a one-step hydromental approach of green luminescent phenol formaldehyde resin (PFR) nanoparticles with the characteristic of stable luminescence and good monodispersibility (Guo et al., 2008; Yang et al., 2012a; Yang et al., 2012b). Moreover, PFR nanoparticles are low toxicity in water with controllable size. So PFR are attracting considerable attention as a promising alternative compared to semiconductor QDs. The PFR are versatile and amenable to surface functionalities, which can be used in a wide range of technologies, such as bioimaging, biosensing and photothermal therapy.
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Scheme 1. Schematic illustration of the design for GSH detection using MnO2-modified PFR.
2. Experimental section
ethanol (ϕf ¼0.74) were used as fluorescence standard (Jones et al., 1980). Fluorescence decay histograms were obtained on an Edinburgh instrument FLS920 spectrometer equipped with a supercontinue white laser (400–700 nm), using the time-correlated single photon counting technique in 4096 channels. Histograms of the instrument response functions (using a LUDOX scatterer) and sample decays were recorded until they typically reached 1.0 104 counts in the peak channel. The monitored wavelengths were 490 nm, 510 nm, and 530 nm. Obtained histograms were fitted as sums of the exponentials, using Gaussian-weighted nonlinear least squares fitting based on Marquardt–Levenberg minimization implemented in the software package of the instrument. The fitting parameters (decay times and pre-exponential factors) were determined by minimizing the reduced χ2. An additional graphical method was used to judge the quality of the fit that included plots of surfaces (‘‘carpets’’) of the weighted residuals νs. channel number. All curve fittings presented here had χ2 values o1.2.
2.1. Chemicals
2.4. Synthesis of PFR
Phenol and KMnO4 were purchased from Guangfu Reagent Company (Tianjin, China). Hexamethylene tetraamine (HMT) was purchased from Sinopharm chemical Regent Co. Ltd (Beijing, China). 4-Morpholineethane sulfonic acid (MES) was purchased from Energy chemical (Shanghai, China). L-glutathione, Bovine serum albumin (BSA), D-fructose, cysteine (Cys), ascorbic acid (AA), homocysteine (Hcys), glutamic acid (Glu), glycine (Gly) and D-aspartic acid (Asp) were purchased from heowns (Tianjing, China). Ultrapure water obtained from a Milli-Q ultrapure (18.2 MΩ cm) system was used in all experiments.
PFR NPs were synthesized according to a reported method (Guo et al., 2008). In a typical synthesis, 0.05 mmol phenol (P) and 0.025 mmol HMT were dissolved in 22 mL of water. After stirring gently for about ten minutes, the solution was transferred into a Teflon-lined autoclave of 30 mL and heated to 160 °C for 1.5 h. A solution with yellow to light yellow and grey color was finally obtained. These products were washed several times by deionized water and pure ethanol to remove inorganic ions and other impurities.
2.2. Instrument
In a typical reaction, 750 μL of PFR was added to 2.5 mL of MES buffer (0.1 M, pH 6.0). One milliliter (1 mL) of KMnO4 (10 mM) was then added and the volume of mixture was adjusted to 10 mL with ultrapure water. After that, the resulting mixture was sonicated for 30 min until a brown colloid was formed. Subsequently, the MnO2PFR nanocomposite was collected by centrifugation and washed three times with ultrapure water to remove the excess potassium and free manganese ions. The product then was redispersed in 10 mL of ultrapure water.
In this paper, we report a novel nanoprobe based on MnO2modified PFR for rapid and selective sensing of GSH. The design and working mechanism of our nanoprobe is suggested in Scheme 1. MnO2-PFR nanocomposite was fabricated through in situ synthesis of MnO2 nanosheets in PFR colloidal solutions. As soon as MnO2-PFR nanocomposite was formed, the fluorescence of PFR in this nanocomposite can be effectively quenched, due to the distance between PFR and MnO2 nanosheets is close enough and consequently FRET happened. Interestingly, this MnO2-induced quenching effect could be recovered in the presence of GSH because of the decomposition of the MnO2 to Mn2 þ by GSH. Moreover, the nanocomposites also exhibit high selectivity towards GSH relative to other biomolecules and electrolytes. Finally, the practical use of this nanoprobe for GSH determination in human serum samples was also presented.
The transmission electron microscopy (TEM) was taken on a JEOL JEM2100 TEM instrument at an accelerating voltage of 200 keV. FT-IR spectra were conducted within the 4000–400 cm 1 wavenumber range using a Nicolet 360 FTIR spectrometer with the KBr pellet technique. X-ray photoelectron spectra (XPS) was measured on a PHI-550 spectrometer by using Mg Ka radiation (hn ¼ 1253.6 eV) photoemission spectroscopy with a base vacuum operated at 300 W. The hydrodynamic diameter and size distribution was measured with dynamic scattered light (BI-200SM) at room temperature using water as the solvent. 2.3. UV–vis absorption and steady-state and time-resolved fluorescence spectroscopy UV–vis absorption spectra were recorded on a Varian UVCary100 spectrophotometer, and for the corrected steady-state fluorescence emission spectra, a FLS920 spectrofluorometer was employed. For the determination of fluorescence quantum yields ϕf, only dilute solutions with the absorbance below 0.1 at the excitation wavelength (λex ¼400 nm) were used. Coumarin 102 in
2.5. Synthesis of MnO2-PFR nanocomposite
2.6. Fluorescence sensing of GSH For the GSH detection, the sensing solutions were prepared by mixing 10 μL of MnO2-PFR nanocomposite solutions with 10 μL of different concentrations of GSH in 1.5 mL centrifuge tubes at room temperature. After the incubation of the required reaction time, the solutions were diluted to 250 μL in ultrapure water and mixed thoroughly. Afterward, the mixtures were transferred into a 1 cm quartz cuvette and the fluorescence emission spectra were measured with excitation at 400 nm. The kinetic behaviors were studied by monitoring the fluorescence recovery of MnO2- PFR nanocomposite by GSH in the different incubation time.
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2.7. Sensing of GSH in human serum samples Human serum samples (provided by the Hospital of Lanzhou University) were treated by centrifugation at 10,000 rpm for 10 min. The supernatant was diluted 20-fold, and mixed with GSH (20, 30 and 40 μM) in 3 mL centrifuge tube for MnO2-PFR nanocomposite. Then, solution was measured after incubation at room temperature for 5 min. Finally, fluorescence intensities at a wavelength of 507 nm were recorded.
3. Result and discussion 3.1. Preparation and characterization of MnO2-PFR nanocomposite As a starting point view of our study, the PFR were prepared by a simple, economic, one-step hydrothermal reaction of phenol and hexamethylenetetramine (HMT). PFRs are highly water dispersible
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due to the hydroxyl and amino group on the surface of PFR. MnO2 nanosheets grew on the surface of the as-synthesized PFR by addition of an aqueous KMnO4 solution in the presence of 2-(N-morpholino) ethanesulfonic acid (MES) buffer at pH 6. MnO2 nanosheets were synthesized by reducing KMnO4 in MES buffer. MnO2-PFR nanocomposite was characterized by TEM, FTIR and XPS. The morphology of the PFR, MnO2 nanosheet and MnO2-PFR was characterized by transmission electron microscopy (TEM). The TEM illustrates that the PFR are spherical and well dispersed, and the average diameter is about 200–400 nm (Fig. 1a). Fig. 1b shows the MnO2 nanosheet with a large 2D and ultrathin plane with occasional folds and wrinkles. Fig. 1c shows that spherical PFR loaded on wrinkle MnO2 nanosheet. Importantly, the large surface area of MnO2 nanosheet provides an essential platform for reacting with a trace of GSH and eventually benefits the high sensitivity of the probe. Dynamic light scattering (DLS) (Fig. 1d) analysis also shows the average hydrodynamic diameter of PFR is about 300– 500 nm.
Fig. 1. TEM image of (a) PFR, (b) MnO2 nanosheet , (c) MnO2-PFR nanocomposite, (d) DLS of PFR, (e) FT-IR spectra of (curve a) PFR, (curve b) MnO2 nanosheet and (curve c) MnO2-PFR nanocomposite and (f) XPS spectra of PFR (curve a) and MnO2-PFR nanocomposite (curve b) in the wide scan survey.
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FT-IR and X-ray photoelectron spectroscopy (XPS) further provide the information of the successful deposition of the PFR on the surface of MnO2 nanosheet. The FTIR spectrum of the PFR, MnO2 and MnO2-PFR nanocomposite are given in Fig. 1e. From Fig. 1e (curve a), the band at 3370 cm 1 originates from phenolic O–H stretching, while the band at 1360 cm 1 is assigned to O–H inplane stretching. The peaks at 2830 cm 1 correspond to aliphatic C–H vibration, and the peaks at 1633, 1600 and 1500 cm 1 are attributed to the C ¼C stretching of aromatic ring. In addition, the bands at 1233 and 1126 cm 1 are due to aromatic C–O stretching. As shown in Fig. 1e (curve b), the broad band of MnO2 at 3373 cm 1 should be attributed to the O–H stretching vibration, and 1643, 1457 and 1174 cm 1 bands are normally attributed to O–H bending vibrations. In the low frequency region, bands around 516.3 cm 1 should be ascribed to the Mn–O and Mn–O– Mn vibrations in [MnO6] octahedral (Chu et al., 2010). From the comparison, it could be observed that the MnO2-PFR nanocomposite (Fig. 1e (curve c)) contained the characteristic peaks of both as-prepared PFR and MnO2, PFR are attached to the surface of the MnO2 nanosheets. Fig. 1f shows the X-ray photoelectron spectroscopy (XPS) of PFR and MnO2-PFR nanocomposite. It can be observed that the spectroscopy of PFR (Fig. 1f (curve a)) shows the existence of carbon (C 1s, 285.1 eV), nitrogen (N 1s, 399.1 eV), oxygen (O 1s, 532.1 eV). Compared to PFR, the presence of the peak at 642.6eV which was observed in the spectra of the MnO2PFR nanocomposite (Fig. 1f (curve b)) was attributed to Mn2p,
respectively. Obviously, these reports confirmed that the successful preparation MnO2-PFR nanocomposite. The optical properties of PFR and MnO2 nanosheets were investigated. The absorption spectrum of PFR (Fig. 2a) exhibits bands at around 258 nm, 312 nm and 392 nm. Under the excitation at 400 nm, the PFR exhibits a bright green emission at around 507 nm. Fig. 2b shows the normalized UV–vis absorption of MnO2 nanosheets and normalized fluorescence emission spectra of PFR. The absorption spectra of MnO2 nanosheets is generally characterized by the expected a broad absorption bands from 235 nm to 740 nm. The absorption band of MnO2 nanosheets overlaps well with the fluorescence emission (507 nm) of PFR, thereby enabling Fö rster resonance energy transfer. The energy transfer from PFR to MnO2 nanosheets leads to the 507 nm emission band was strongly quenched by MnO2 nanosheets. The fluorescence emission spectra of PFR as a function of [KMnO4] (MnO2 nanosheets were prepared by reducing KMnO4) as shown in Fig. S1. Upon increasing the KMnO4 concentration, a large decrease of the fluorescence intensity and fluorescence quantum yield was observed. The spectral change results in a clear color change from yellow to brown, when KMnO4 is added to the water solution of PFR (Inset of Fig. S1a).When the concentration of MnO2 nanosheets was higher than 0.9 mM, the maximum fluorescence quenching degree of the MnO2-PFR nanocomposite (Fig. S1a) is up to 96%. The quantum yield of PFR decreases from 4.0% in the absence of KMnO4 to 0.12% in addition of KMnO4 up to 0.9 mM.
Fig. 2. (a) Normalized UV–vis (solid) and steady-state fluorescence spectra (dash) of PFR in water. Inset shows the PFR under daylight and UV light in water solution. (b) Spectral overlap showing the UV–vis absorption spectra of MnO2 nanosheets (solid) and the fluorescence emission spectra (dash) of the PFR in water.
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attributed to the reduction of MnO2 to Mn2 þ , which is based on the unique reaction between GSH and MnO2 nanosheet. During this redox reaction, GSH was oxidized to generate glutathione disulfide (GSSG) through thiol-disufide exchange as shown in Eq. (1). MnO2–PFRþGSH þ2H þ -Mn2 þ þPFRþGSSG þ2H2O
(1)
Fluorescence emission spectra of MnO2-PFR nanoposites measured as a function of GSH concentration are shown in Fig. 3a. The fluorescence intensity at 507 nm of the sensing system continuously increased with sequential addition of GSH under the excitation of 400 nm. The restored fluorescence was dependent on the amount of GSH. When the concentration of GSH was increased to 900 μM, no further restoring of fluorescence can be observed, showing that the sensing response has reached the maximum. As shown in Fig. 3b, the fluorescence intensity increased almost linearly with increasing GSH concentration (0–100 μM). The calibration curve (Fig. 3c) for GSH concentrations was obtained as shown below, where ΔF stands for the fluorescence enhancement against the concentration of GSH.
ΔF = 1592 + 72.97 × [GSH ] (r = 0.9942, [GSH ] = 0 − 100 μM) The calibration equation serves as the quantitative basis for the detection of GSH content in water. The detection limit (DL) was estimated based on the following equation: DL ¼3.3s/k, where s is the standard deviation of the blank sample and k is the slope of the calibration curve. The DL of MnO2-PFR nanocomposite was 7.6 nM. As shown in Table S1, the detection limit obtained from this method was found to be higher or comparable than those of the others. The result indicated that the MnO2-PFR nanocomposite could be potentially utilized as an excellent optical sensor for the quantitative analysis of GSH in biological samples. To investigate the fluorescence dynamics of PFR, fluorescence decay traces of PFR was recorded at three emission wavelengths (490 nm, 510 nm and 530 nm) by the single-photon timing method (Boens et al., 2007). Fluorescence decay for PFR water revealed bi-exponential behavior. The fluorescence decay is fitted to the bi-exponential profile with the lifetime of 3.5 ns and 1.5 ns in water in the all fluorescence region. The multiexponential nature of the lifetime suggests that the components of PFR in water are complicated, probably due to the involvement of different particle sizes and emissive trap sites. Upon addition of 0.9 mM of KMnO4 to water solution of PFR,
Fig. 3. (a) Fluorescence emission spectra (λex ¼ 400 nm) of MnO2-PFR nanocomposite in the presence of different concentrations of GSH (0–900 μM) (b) Relationship between fluorescence enhancement and the target concentration. (c) Calibration curves between relative fluorescence intensity of MnO2-PFR and GSH concentration. F0 represents the fluorescence intensity of MnO2-PFR without GSH, and F represents the fluorescence intensity with different GSH concentration (λex ¼ 400 nm; λem ¼507 nm).
As shown in Fig. S1b, the fluorescence intensity decreased almost linearly with increasing KMnO4 concentration (r ¼ 0.99). 3.2. Fluorescence sensing of GSH in an aqueous solution with the MnO2-PFR nanocomposite MnO2 in the proximity of PFR rendered the fluorescence of PFR in the OFF state due to an efficient FRET process. However, with the addition of GSH the fluorescence enhancement of PFR could be
Fig. 4. Selectivity of the MnO2-PFR nanocomposite for GSH over other potential interferences (0.1 M each; for bovine serum albumin: 1 mg/ml). (a) KCl, (b) NaCl, (c) MgSO4, (d) CoCl2, (e) NaSO4, (f) MnCl2, (g) CaCl2, (h) Tris 7.0, (i) HEPES, (j) PBS, (k) BSA, (l) Glu, (m) Gly, (n) Asp, (o) fructose, (p) ascorbic acid, (q) HCys, (r) Cys and (s) GSH. F0 and F are the fluorescence intensity of the probe in the absence and presence of the target (GSH) or nontarget samples, respectively.
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Table 1 Determination results of GSH in diluted human serum. Found in sample (μM) n ¼5
Added (μM)
Total found (μM) Recovery (%) n¼ 5
RSD (%) n ¼5
28.357 2.25
20.00 30.00 40.00
49.077 2.94 57.517 4.09 70.39 7 5.62
103.6 97.2 105.1
4.8 5.4 6.2
24.287 2.37
20.00 30.00 40.00
45.78 7 3.46 54.047 4.37 63.84 7 5.18
107.5 99.2 98.9
5.6 5.9 6.1
31.18 7 2.14
20.00 30.00 40.00
50.52 7 4.48 62.05 7 5.02 69.30 7 6.36
96.7 102.9 95.3
6.1 6.3 6.6
30.777 2.37
20.00 30.00 40.00
51.99 7 4.21 61.85 7 5.77 71.377 5.87
106.1 103.6 101.5
5.8 6.5 5.9
22.23 7 2.40
20.00 30.00 40.00
41.497 3.69 51.87 7 4.52 63.317 5.27
96.3 98.8 102.7
6.4 6.3 6.0
26.30 7 2.21
20.00 30.00 40.00
47.067 3.13 55.047 5.08 66.147 5.67
103.8 95.8 99.6
4.9 6.5 6.2
the fluorescence decay time does not change (Fig. S2). The measured lifetime in the presence of KMnO4 is the same as the lifetime of the PFR in water. With the further increase of KMnO4 concentration, the fluorescence decay time does not change either. A possible explanation for the fluorescence emission and lifetime changes of PFR with KMnO4 is the FRET process. As indicated in Fig. 2b, the absorption band of MnO2 nanosheets overlaps well with the fluorescence emission of PFR, Consequently, in the presence of KMnO4 (MnO2 nanosheets were prepared by reducing KMnO4), the FRET from the PFR (emission at about 507 nm) to the non-fluorescent MnO2 nanosheets is enhanced. This enhancement in the FRET induces quenching of the fluorescence as a function of KMnO4 concentration. The decrease of the fluorescence intensity with the increase the concentration of KMnO4 means that the concentration of the nonfluorescent MnO2-PFR nanocomposite increases. The non-fluorescent MnO2-PFR nanocomposite doses not change the fluorescence emission maximum wavelength and the lifetime of the PFR. To better explore the quenching mechanism, we also calculated the quenching equation according to the Stern–Volmer equation F0/F¼1 þ Ks[Q] from Fig. S1b, where Ks is the association equilibrium constant, F0 is the initial fluorescence intensity without the quencher (KMnO4) , F is the fluorescence intensity after adding the quencher of concentration. As shown in Fig. S1b, a good linear Stern–Volmer relationship is observed in PFR for KMnO4. Since the presence of KMnO4 in the PFR water solution does not change the fluorescence the lifetime of PFR, we may conclude that the quenching process is a static mechanism. 3.3. Selectivity of PFR MnO2 nanocomposite-based turn-on fluorescence toward GSH The selectivity of MnO2-PFR nanocomposite toward GSH was evaluated by screening its response to biological ions and amino acids. As shown in Fig. 4, the fluorescence intensity of MnO2-PFR nanocomposite displays a dramatic increase in the presence of GSH. In contrast, no obvious increase in fluorescent intensity could
be observed by adding biological ions and amino acids. Cysteine (Cys), homocysteine (HCys), fructose and ascorbic acid can cause the fluorescent response to MnO2-PFR nanocomposite. However, the content of these interferents in biological system is much lower than GSH (Jacobsen et al., 1994; Michelet et al., 1995). Hence, the MnO2-PFR nanocomposite can be used as a selective fluorescent nanoprobe for the light-on detection of the human serum GSH without significant interference from small molecular thiols. 3.4. Determination of GSH concentration in human serum To demonstrate the application of the developed MnO2-PFR nanocomposite nanosensor in complicated biological environment, we next used human serum to detect the GSH concentration. The accuracy of the sample analysis was tested by spiking a known amount of standard GSH with the clinical human serum samples which were donated by Lan zhou University Hospital (Lan zhou, China) and calculating its recovery, and then was added into the MnO2-PFR solution. The mixture was incubated at room temperature for 5 min and then monitored. As shown in Table 1, it can be seen that, the average original GSH concentration in the human serum was 26.71 7 4.47 μM. The obtained GSH concentration ranges in human blood were in agreement with reports in the literature (Michelet et al., 1995). Furthermore, recoveries of different known amounts of added GSH were obtained from 95.3% to 107.5%. The good recovery showed that MnO2-PFR nanocomposite possessed the feasibility and reliability for real clinical sample analysis. Overall, these findings demonstrated that MnO2PFR nanocomposite is suitable to detect GSH in real biological environments.
4. Conclusion We have developed a new MnO2-PFR nanocomposite nanoprobe for fluorescence “turn-on” detection of GSH and can be use for detecting glutathione in blood serum. The PFR were served as the fluorescence units and the MnO2 nanosheets were used as the quencher. When MnO2 was assembled on the surface of the PFR, the fluorescence of the PFR can be efficiently quenched by the MnO2. In the existence of GSH, MnO2 was reduced to Mn2 þ and the fluorescence of PFR can be recovered. Moreover, it exhibits remarkable fluorescence enhancement to GSH, which affords a high sensitivity to GSH in aqueous solutions with a detection limit of 7.6 nM observed. In the actual sample analysis, the good recoveries ranging from 95.3% to 107.5% illustrated that the application of the MnO2-PFR nanocomposite in biological sample analysis was anticipated to be promising.
Acknowledgments The authors would like to thank the Natural Science Foundation of China (No. 21271094). This work was supported by the National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China (Grant no. J1103307).
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.2015.09.044.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Fluorescent glutathione probe based on MnO2-phenol formaldehyde resin nanocomposite Xudong Wang a, Dan Wang a, Yali Guo a, Chengduan Yang a, Xiaoyu Liu a, Anam Iqbal a, Weisheng Liu a, Wenwu Qin a,n, Dan Yan b, Huichen Guo b,n a Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province and State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, PR China b State Key Laboratory of Veterinary Etiological Biology and Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 1, Lanzhou, Gansu 730046, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 17 July 2015 Received in revised form 10 September 2015 Accepted 19 September 2015 Available online 25 September 2015
MnO2-phenol formaldehyde resin (MnO2-PFR) nanocomposite is successfully prepared by a simple chemical reduction process. The resultant MnO2-PFR nanocomposite is well characterized. The absorption band of non-fluorescent MnO2 nanosheets overlaps well with the fluorescence emission of PFR nanoparticles. The green fluorescence of PFR in this nanocomposite can be effectively quenched by fluorescence resonance energy transfer from PFR to MnO2. In the presence of glutathione (GSH), the fluorescence of PFR could be recovered due to MnO2 was reduced to Mn2 þ by GSH. The nanocomposite can be use for detecting glutathione in blood serum. & 2015 Elsevier B.V. All rights reserved.
Keywords: MnO2 Phenol formaldehyde resin Glutathione
1. Introduction The low-molecular weight thiol glutathione (γ-L-glutamyl-Lcysteinyl-glycine) (GSH) play a central role in maintaining the appropriate oxidation–reduction state of proteins, cells and organisms (Hirrlinger and Dringen, 2010). The oxidative stress has been found in many diseases (including kwashiorkor, seizure, Alzheimers’s disease, Parkinson’s disease, liver disease, cystic fibrosis, sickle cell anemia, HIV, AIDS, cancer, heart attack, stroke and diabetes) and accelerates the aging process (Wu et al., 2004). Thus, it is of great importance to design and develop the analytical strategies for the analysis of biomarkers, of oxidative stress such as GSH. During the past decades, the measurement of GSH is carried out using various detection techniques, including fluorescent spectroscopy, (Kim et al., 2011) electrochemical pulse voltammetric methods, (Safavi et al., 2009) high-performance liquid chromatography (HPLC), (Townsend et al., 2003) and colorimetric assays (Uehara et al., 2010). Among the various analytical methods, the fluorescent spectroscopy offers significant advantages over other techniques due to its generally non-destructive character, its high sensitivity and simplicity. Currently, various fluorescent n
Corresponding authors. Fax: þ 86 931 8912582. E-mail addresses: [email protected] (W. Qin), [email protected] (H. Guo). http://dx.doi.org/10.1016/j.bios.2015.09.044 0956-5663/& 2015 Elsevier B.V. All rights reserved.
probes, using organic fluorophores, (Kim et al., 2011) gold nanoclusters, (Tian et al., 2012) quantum dots (QDs), (Banerjee et al., 2009; Liu et al., 2010; Zhang et al., 2009) carbon nanomaterials, (Cai et al., 2015; Shi et al., 2014; Wang et al., 2015; Zhang et al., 2014; Zhou et al., 2012) and upconversion nanoparticles (UCPs), (Deng et al., 2011) have been developed for GSH sensing. Recently, Santra et al., designed and synthesized a simple quantum-dotbased OFF–ON fluorescent probe for detecting glutathione with high selectivity and sensitivity (Banerjee et al., 2009). However, the toxicity of these QDs has to be remedied if one wants to design fluorescent GSH probes use inside the biological cells due to QDs contain Cd et al heavy metal ions. A sensitive turn-on sensor for GSH in the cell samples based on the recovered fluorescence of the gold nanoclusters Hg2 þ system was reported by Zhu and coworkers (Tian et al., 2012). However, Hg is a kind of wellknown toxicity and potential environmental hazard of the heavy metals. Recently, Yu’s group has reported a one-step hydromental approach of green luminescent phenol formaldehyde resin (PFR) nanoparticles with the characteristic of stable luminescence and good monodispersibility (Guo et al., 2008; Yang et al., 2012a; Yang et al., 2012b). Moreover, PFR nanoparticles are low toxicity in water with controllable size. So PFR are attracting considerable attention as a promising alternative compared to semiconductor QDs. The PFR are versatile and amenable to surface functionalities, which can be used in a wide range of technologies, such as bioimaging, biosensing and photothermal therapy.
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Scheme 1. Schematic illustration of the design for GSH detection using MnO2-modified PFR.
2. Experimental section
ethanol (ϕf ¼0.74) were used as fluorescence standard (Jones et al., 1980). Fluorescence decay histograms were obtained on an Edinburgh instrument FLS920 spectrometer equipped with a supercontinue white laser (400–700 nm), using the time-correlated single photon counting technique in 4096 channels. Histograms of the instrument response functions (using a LUDOX scatterer) and sample decays were recorded until they typically reached 1.0 104 counts in the peak channel. The monitored wavelengths were 490 nm, 510 nm, and 530 nm. Obtained histograms were fitted as sums of the exponentials, using Gaussian-weighted nonlinear least squares fitting based on Marquardt–Levenberg minimization implemented in the software package of the instrument. The fitting parameters (decay times and pre-exponential factors) were determined by minimizing the reduced χ2. An additional graphical method was used to judge the quality of the fit that included plots of surfaces (‘‘carpets’’) of the weighted residuals νs. channel number. All curve fittings presented here had χ2 values o1.2.
2.1. Chemicals
2.4. Synthesis of PFR
Phenol and KMnO4 were purchased from Guangfu Reagent Company (Tianjin, China). Hexamethylene tetraamine (HMT) was purchased from Sinopharm chemical Regent Co. Ltd (Beijing, China). 4-Morpholineethane sulfonic acid (MES) was purchased from Energy chemical (Shanghai, China). L-glutathione, Bovine serum albumin (BSA), D-fructose, cysteine (Cys), ascorbic acid (AA), homocysteine (Hcys), glutamic acid (Glu), glycine (Gly) and D-aspartic acid (Asp) were purchased from heowns (Tianjing, China). Ultrapure water obtained from a Milli-Q ultrapure (18.2 MΩ cm) system was used in all experiments.
PFR NPs were synthesized according to a reported method (Guo et al., 2008). In a typical synthesis, 0.05 mmol phenol (P) and 0.025 mmol HMT were dissolved in 22 mL of water. After stirring gently for about ten minutes, the solution was transferred into a Teflon-lined autoclave of 30 mL and heated to 160 °C for 1.5 h. A solution with yellow to light yellow and grey color was finally obtained. These products were washed several times by deionized water and pure ethanol to remove inorganic ions and other impurities.
2.2. Instrument
In a typical reaction, 750 μL of PFR was added to 2.5 mL of MES buffer (0.1 M, pH 6.0). One milliliter (1 mL) of KMnO4 (10 mM) was then added and the volume of mixture was adjusted to 10 mL with ultrapure water. After that, the resulting mixture was sonicated for 30 min until a brown colloid was formed. Subsequently, the MnO2PFR nanocomposite was collected by centrifugation and washed three times with ultrapure water to remove the excess potassium and free manganese ions. The product then was redispersed in 10 mL of ultrapure water.
In this paper, we report a novel nanoprobe based on MnO2modified PFR for rapid and selective sensing of GSH. The design and working mechanism of our nanoprobe is suggested in Scheme 1. MnO2-PFR nanocomposite was fabricated through in situ synthesis of MnO2 nanosheets in PFR colloidal solutions. As soon as MnO2-PFR nanocomposite was formed, the fluorescence of PFR in this nanocomposite can be effectively quenched, due to the distance between PFR and MnO2 nanosheets is close enough and consequently FRET happened. Interestingly, this MnO2-induced quenching effect could be recovered in the presence of GSH because of the decomposition of the MnO2 to Mn2 þ by GSH. Moreover, the nanocomposites also exhibit high selectivity towards GSH relative to other biomolecules and electrolytes. Finally, the practical use of this nanoprobe for GSH determination in human serum samples was also presented.
The transmission electron microscopy (TEM) was taken on a JEOL JEM2100 TEM instrument at an accelerating voltage of 200 keV. FT-IR spectra were conducted within the 4000–400 cm 1 wavenumber range using a Nicolet 360 FTIR spectrometer with the KBr pellet technique. X-ray photoelectron spectra (XPS) was measured on a PHI-550 spectrometer by using Mg Ka radiation (hn ¼ 1253.6 eV) photoemission spectroscopy with a base vacuum operated at 300 W. The hydrodynamic diameter and size distribution was measured with dynamic scattered light (BI-200SM) at room temperature using water as the solvent. 2.3. UV–vis absorption and steady-state and time-resolved fluorescence spectroscopy UV–vis absorption spectra were recorded on a Varian UVCary100 spectrophotometer, and for the corrected steady-state fluorescence emission spectra, a FLS920 spectrofluorometer was employed. For the determination of fluorescence quantum yields ϕf, only dilute solutions with the absorbance below 0.1 at the excitation wavelength (λex ¼400 nm) were used. Coumarin 102 in
2.5. Synthesis of MnO2-PFR nanocomposite
2.6. Fluorescence sensing of GSH For the GSH detection, the sensing solutions were prepared by mixing 10 μL of MnO2-PFR nanocomposite solutions with 10 μL of different concentrations of GSH in 1.5 mL centrifuge tubes at room temperature. After the incubation of the required reaction time, the solutions were diluted to 250 μL in ultrapure water and mixed thoroughly. Afterward, the mixtures were transferred into a 1 cm quartz cuvette and the fluorescence emission spectra were measured with excitation at 400 nm. The kinetic behaviors were studied by monitoring the fluorescence recovery of MnO2- PFR nanocomposite by GSH in the different incubation time.
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2.7. Sensing of GSH in human serum samples Human serum samples (provided by the Hospital of Lanzhou University) were treated by centrifugation at 10,000 rpm for 10 min. The supernatant was diluted 20-fold, and mixed with GSH (20, 30 and 40 μM) in 3 mL centrifuge tube for MnO2-PFR nanocomposite. Then, solution was measured after incubation at room temperature for 5 min. Finally, fluorescence intensities at a wavelength of 507 nm were recorded.
3. Result and discussion 3.1. Preparation and characterization of MnO2-PFR nanocomposite As a starting point view of our study, the PFR were prepared by a simple, economic, one-step hydrothermal reaction of phenol and hexamethylenetetramine (HMT). PFRs are highly water dispersible
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due to the hydroxyl and amino group on the surface of PFR. MnO2 nanosheets grew on the surface of the as-synthesized PFR by addition of an aqueous KMnO4 solution in the presence of 2-(N-morpholino) ethanesulfonic acid (MES) buffer at pH 6. MnO2 nanosheets were synthesized by reducing KMnO4 in MES buffer. MnO2-PFR nanocomposite was characterized by TEM, FTIR and XPS. The morphology of the PFR, MnO2 nanosheet and MnO2-PFR was characterized by transmission electron microscopy (TEM). The TEM illustrates that the PFR are spherical and well dispersed, and the average diameter is about 200–400 nm (Fig. 1a). Fig. 1b shows the MnO2 nanosheet with a large 2D and ultrathin plane with occasional folds and wrinkles. Fig. 1c shows that spherical PFR loaded on wrinkle MnO2 nanosheet. Importantly, the large surface area of MnO2 nanosheet provides an essential platform for reacting with a trace of GSH and eventually benefits the high sensitivity of the probe. Dynamic light scattering (DLS) (Fig. 1d) analysis also shows the average hydrodynamic diameter of PFR is about 300– 500 nm.
Fig. 1. TEM image of (a) PFR, (b) MnO2 nanosheet , (c) MnO2-PFR nanocomposite, (d) DLS of PFR, (e) FT-IR spectra of (curve a) PFR, (curve b) MnO2 nanosheet and (curve c) MnO2-PFR nanocomposite and (f) XPS spectra of PFR (curve a) and MnO2-PFR nanocomposite (curve b) in the wide scan survey.
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FT-IR and X-ray photoelectron spectroscopy (XPS) further provide the information of the successful deposition of the PFR on the surface of MnO2 nanosheet. The FTIR spectrum of the PFR, MnO2 and MnO2-PFR nanocomposite are given in Fig. 1e. From Fig. 1e (curve a), the band at 3370 cm 1 originates from phenolic O–H stretching, while the band at 1360 cm 1 is assigned to O–H inplane stretching. The peaks at 2830 cm 1 correspond to aliphatic C–H vibration, and the peaks at 1633, 1600 and 1500 cm 1 are attributed to the C ¼C stretching of aromatic ring. In addition, the bands at 1233 and 1126 cm 1 are due to aromatic C–O stretching. As shown in Fig. 1e (curve b), the broad band of MnO2 at 3373 cm 1 should be attributed to the O–H stretching vibration, and 1643, 1457 and 1174 cm 1 bands are normally attributed to O–H bending vibrations. In the low frequency region, bands around 516.3 cm 1 should be ascribed to the Mn–O and Mn–O– Mn vibrations in [MnO6] octahedral (Chu et al., 2010). From the comparison, it could be observed that the MnO2-PFR nanocomposite (Fig. 1e (curve c)) contained the characteristic peaks of both as-prepared PFR and MnO2, PFR are attached to the surface of the MnO2 nanosheets. Fig. 1f shows the X-ray photoelectron spectroscopy (XPS) of PFR and MnO2-PFR nanocomposite. It can be observed that the spectroscopy of PFR (Fig. 1f (curve a)) shows the existence of carbon (C 1s, 285.1 eV), nitrogen (N 1s, 399.1 eV), oxygen (O 1s, 532.1 eV). Compared to PFR, the presence of the peak at 642.6eV which was observed in the spectra of the MnO2PFR nanocomposite (Fig. 1f (curve b)) was attributed to Mn2p,
respectively. Obviously, these reports confirmed that the successful preparation MnO2-PFR nanocomposite. The optical properties of PFR and MnO2 nanosheets were investigated. The absorption spectrum of PFR (Fig. 2a) exhibits bands at around 258 nm, 312 nm and 392 nm. Under the excitation at 400 nm, the PFR exhibits a bright green emission at around 507 nm. Fig. 2b shows the normalized UV–vis absorption of MnO2 nanosheets and normalized fluorescence emission spectra of PFR. The absorption spectra of MnO2 nanosheets is generally characterized by the expected a broad absorption bands from 235 nm to 740 nm. The absorption band of MnO2 nanosheets overlaps well with the fluorescence emission (507 nm) of PFR, thereby enabling Fö rster resonance energy transfer. The energy transfer from PFR to MnO2 nanosheets leads to the 507 nm emission band was strongly quenched by MnO2 nanosheets. The fluorescence emission spectra of PFR as a function of [KMnO4] (MnO2 nanosheets were prepared by reducing KMnO4) as shown in Fig. S1. Upon increasing the KMnO4 concentration, a large decrease of the fluorescence intensity and fluorescence quantum yield was observed. The spectral change results in a clear color change from yellow to brown, when KMnO4 is added to the water solution of PFR (Inset of Fig. S1a).When the concentration of MnO2 nanosheets was higher than 0.9 mM, the maximum fluorescence quenching degree of the MnO2-PFR nanocomposite (Fig. S1a) is up to 96%. The quantum yield of PFR decreases from 4.0% in the absence of KMnO4 to 0.12% in addition of KMnO4 up to 0.9 mM.
Fig. 2. (a) Normalized UV–vis (solid) and steady-state fluorescence spectra (dash) of PFR in water. Inset shows the PFR under daylight and UV light in water solution. (b) Spectral overlap showing the UV–vis absorption spectra of MnO2 nanosheets (solid) and the fluorescence emission spectra (dash) of the PFR in water.
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attributed to the reduction of MnO2 to Mn2 þ , which is based on the unique reaction between GSH and MnO2 nanosheet. During this redox reaction, GSH was oxidized to generate glutathione disulfide (GSSG) through thiol-disufide exchange as shown in Eq. (1). MnO2–PFRþGSH þ2H þ -Mn2 þ þPFRþGSSG þ2H2O
(1)
Fluorescence emission spectra of MnO2-PFR nanoposites measured as a function of GSH concentration are shown in Fig. 3a. The fluorescence intensity at 507 nm of the sensing system continuously increased with sequential addition of GSH under the excitation of 400 nm. The restored fluorescence was dependent on the amount of GSH. When the concentration of GSH was increased to 900 μM, no further restoring of fluorescence can be observed, showing that the sensing response has reached the maximum. As shown in Fig. 3b, the fluorescence intensity increased almost linearly with increasing GSH concentration (0–100 μM). The calibration curve (Fig. 3c) for GSH concentrations was obtained as shown below, where ΔF stands for the fluorescence enhancement against the concentration of GSH.
ΔF = 1592 + 72.97 × [GSH ] (r = 0.9942, [GSH ] = 0 − 100 μM) The calibration equation serves as the quantitative basis for the detection of GSH content in water. The detection limit (DL) was estimated based on the following equation: DL ¼3.3s/k, where s is the standard deviation of the blank sample and k is the slope of the calibration curve. The DL of MnO2-PFR nanocomposite was 7.6 nM. As shown in Table S1, the detection limit obtained from this method was found to be higher or comparable than those of the others. The result indicated that the MnO2-PFR nanocomposite could be potentially utilized as an excellent optical sensor for the quantitative analysis of GSH in biological samples. To investigate the fluorescence dynamics of PFR, fluorescence decay traces of PFR was recorded at three emission wavelengths (490 nm, 510 nm and 530 nm) by the single-photon timing method (Boens et al., 2007). Fluorescence decay for PFR water revealed bi-exponential behavior. The fluorescence decay is fitted to the bi-exponential profile with the lifetime of 3.5 ns and 1.5 ns in water in the all fluorescence region. The multiexponential nature of the lifetime suggests that the components of PFR in water are complicated, probably due to the involvement of different particle sizes and emissive trap sites. Upon addition of 0.9 mM of KMnO4 to water solution of PFR,
Fig. 3. (a) Fluorescence emission spectra (λex ¼ 400 nm) of MnO2-PFR nanocomposite in the presence of different concentrations of GSH (0–900 μM) (b) Relationship between fluorescence enhancement and the target concentration. (c) Calibration curves between relative fluorescence intensity of MnO2-PFR and GSH concentration. F0 represents the fluorescence intensity of MnO2-PFR without GSH, and F represents the fluorescence intensity with different GSH concentration (λex ¼ 400 nm; λem ¼507 nm).
As shown in Fig. S1b, the fluorescence intensity decreased almost linearly with increasing KMnO4 concentration (r ¼ 0.99). 3.2. Fluorescence sensing of GSH in an aqueous solution with the MnO2-PFR nanocomposite MnO2 in the proximity of PFR rendered the fluorescence of PFR in the OFF state due to an efficient FRET process. However, with the addition of GSH the fluorescence enhancement of PFR could be
Fig. 4. Selectivity of the MnO2-PFR nanocomposite for GSH over other potential interferences (0.1 M each; for bovine serum albumin: 1 mg/ml). (a) KCl, (b) NaCl, (c) MgSO4, (d) CoCl2, (e) NaSO4, (f) MnCl2, (g) CaCl2, (h) Tris 7.0, (i) HEPES, (j) PBS, (k) BSA, (l) Glu, (m) Gly, (n) Asp, (o) fructose, (p) ascorbic acid, (q) HCys, (r) Cys and (s) GSH. F0 and F are the fluorescence intensity of the probe in the absence and presence of the target (GSH) or nontarget samples, respectively.
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Table 1 Determination results of GSH in diluted human serum. Found in sample (μM) n ¼5
Added (μM)
Total found (μM) Recovery (%) n¼ 5
RSD (%) n ¼5
28.357 2.25
20.00 30.00 40.00
49.077 2.94 57.517 4.09 70.39 7 5.62
103.6 97.2 105.1
4.8 5.4 6.2
24.287 2.37
20.00 30.00 40.00
45.78 7 3.46 54.047 4.37 63.84 7 5.18
107.5 99.2 98.9
5.6 5.9 6.1
31.18 7 2.14
20.00 30.00 40.00
50.52 7 4.48 62.05 7 5.02 69.30 7 6.36
96.7 102.9 95.3
6.1 6.3 6.6
30.777 2.37
20.00 30.00 40.00
51.99 7 4.21 61.85 7 5.77 71.377 5.87
106.1 103.6 101.5
5.8 6.5 5.9
22.23 7 2.40
20.00 30.00 40.00
41.497 3.69 51.87 7 4.52 63.317 5.27
96.3 98.8 102.7
6.4 6.3 6.0
26.30 7 2.21
20.00 30.00 40.00
47.067 3.13 55.047 5.08 66.147 5.67
103.8 95.8 99.6
4.9 6.5 6.2
the fluorescence decay time does not change (Fig. S2). The measured lifetime in the presence of KMnO4 is the same as the lifetime of the PFR in water. With the further increase of KMnO4 concentration, the fluorescence decay time does not change either. A possible explanation for the fluorescence emission and lifetime changes of PFR with KMnO4 is the FRET process. As indicated in Fig. 2b, the absorption band of MnO2 nanosheets overlaps well with the fluorescence emission of PFR, Consequently, in the presence of KMnO4 (MnO2 nanosheets were prepared by reducing KMnO4), the FRET from the PFR (emission at about 507 nm) to the non-fluorescent MnO2 nanosheets is enhanced. This enhancement in the FRET induces quenching of the fluorescence as a function of KMnO4 concentration. The decrease of the fluorescence intensity with the increase the concentration of KMnO4 means that the concentration of the nonfluorescent MnO2-PFR nanocomposite increases. The non-fluorescent MnO2-PFR nanocomposite doses not change the fluorescence emission maximum wavelength and the lifetime of the PFR. To better explore the quenching mechanism, we also calculated the quenching equation according to the Stern–Volmer equation F0/F¼1 þ Ks[Q] from Fig. S1b, where Ks is the association equilibrium constant, F0 is the initial fluorescence intensity without the quencher (KMnO4) , F is the fluorescence intensity after adding the quencher of concentration. As shown in Fig. S1b, a good linear Stern–Volmer relationship is observed in PFR for KMnO4. Since the presence of KMnO4 in the PFR water solution does not change the fluorescence the lifetime of PFR, we may conclude that the quenching process is a static mechanism. 3.3. Selectivity of PFR MnO2 nanocomposite-based turn-on fluorescence toward GSH The selectivity of MnO2-PFR nanocomposite toward GSH was evaluated by screening its response to biological ions and amino acids. As shown in Fig. 4, the fluorescence intensity of MnO2-PFR nanocomposite displays a dramatic increase in the presence of GSH. In contrast, no obvious increase in fluorescent intensity could
be observed by adding biological ions and amino acids. Cysteine (Cys), homocysteine (HCys), fructose and ascorbic acid can cause the fluorescent response to MnO2-PFR nanocomposite. However, the content of these interferents in biological system is much lower than GSH (Jacobsen et al., 1994; Michelet et al., 1995). Hence, the MnO2-PFR nanocomposite can be used as a selective fluorescent nanoprobe for the light-on detection of the human serum GSH without significant interference from small molecular thiols. 3.4. Determination of GSH concentration in human serum To demonstrate the application of the developed MnO2-PFR nanocomposite nanosensor in complicated biological environment, we next used human serum to detect the GSH concentration. The accuracy of the sample analysis was tested by spiking a known amount of standard GSH with the clinical human serum samples which were donated by Lan zhou University Hospital (Lan zhou, China) and calculating its recovery, and then was added into the MnO2-PFR solution. The mixture was incubated at room temperature for 5 min and then monitored. As shown in Table 1, it can be seen that, the average original GSH concentration in the human serum was 26.71 7 4.47 μM. The obtained GSH concentration ranges in human blood were in agreement with reports in the literature (Michelet et al., 1995). Furthermore, recoveries of different known amounts of added GSH were obtained from 95.3% to 107.5%. The good recovery showed that MnO2-PFR nanocomposite possessed the feasibility and reliability for real clinical sample analysis. Overall, these findings demonstrated that MnO2PFR nanocomposite is suitable to detect GSH in real biological environments.
4. Conclusion We have developed a new MnO2-PFR nanocomposite nanoprobe for fluorescence “turn-on” detection of GSH and can be use for detecting glutathione in blood serum. The PFR were served as the fluorescence units and the MnO2 nanosheets were used as the quencher. When MnO2 was assembled on the surface of the PFR, the fluorescence of the PFR can be efficiently quenched by the MnO2. In the existence of GSH, MnO2 was reduced to Mn2 þ and the fluorescence of PFR can be recovered. Moreover, it exhibits remarkable fluorescence enhancement to GSH, which affords a high sensitivity to GSH in aqueous solutions with a detection limit of 7.6 nM observed. In the actual sample analysis, the good recoveries ranging from 95.3% to 107.5% illustrated that the application of the MnO2-PFR nanocomposite in biological sample analysis was anticipated to be promising.
Acknowledgments The authors would like to thank the Natural Science Foundation of China (No. 21271094). This work was supported by the National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China (Grant no. J1103307).
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.2015.09.044.
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