Author’s Accepted Manuscript A rapid fluorescence “switch-on” assay for Glutathione detection by using carbon Dots-MnO2 nanocomposites Qi-Yong Cai, Jie Li, Jia Ge, Lin Zhang, Ya-Lei Hu, Zhao-Hui Li, Ling-Bo Qu www.elsevier.com/locate/bios
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S0956-5663(15)30077-4 http://dx.doi.org/10.1016/j.bios.2015.04.077 BIOS7645
To appear in: Biosensors and Bioelectronic Received date: 3 February 2015 Revised date: 12 April 2015 Accepted date: 23 April 2015 Cite this article as: Qi-Yong Cai, Jie Li, Jia Ge, Lin Zhang, Ya-Lei Hu, ZhaoHui Li and Ling-Bo Qu, A rapid fluorescence “switch-on” assay for Glutathione detection by using carbon Dots-MnO2 nanocomposites, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.04.077 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A Rapid Fluorescence “Switch-On” Assay for Glutathione Detection by using Carbon Dots-MnO2 Nanocomposites Qi-Yong Caia,1, Jie Lia,1, Jia Gea,b, Lin Zhanga, Ya-Lei Hua, Zhao-Hui Lia,b,*, and Ling-Bo Qua,c,*
a
College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou
450001, P. R. China b
State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University,
Changsha 410082, P.R. China c
School of Chemistry & Chemical Engineering, Henan University of Technology, Zhengzhou 450001, P.R. China
*
Corresponding authors. Tel.: +86-371-67783126; Fax: +86-371-67781556.
E-mail address: [email protected] (Z. H. Li), [email protected] (L. B. Qu). 1 These authors contributed equally to this work. Abstract Glutathione (GSH) serves many cellular functions and plays crucial roles in human pathologies. Simple and sensitive sensors capable of detecting GSH would be useful tools to understand the mechanism of diseases. In this work, a rapid fluorescence “switch-on” assay was developed to detect trace amount of GSH based on carbon dots-MnO2 nanocomposites, which was fabricated through in situ synthesis of MnO2 nanosheets in carbon dots colloid solution. Due to the formation of carbon dots-MnO2 nanocomposites, fluorescence of carbon dots could be quenched efficiently by MnO2 nanosheeets through fluorescence resonance energy transfer (FRET). However, the presence of GSH would reduce MnO2 nanosheets to Mn2+ ions and subsequently release
carbon dots, which resulted in sufficient recovery of fluorescent signal. This proposed assay demonstrated highly selectivity toward GSH with a detection limit of 300 nM. Moreover, this method has also shown sensitive responses to GSH in human serum samples, which indicated its great potential to be used in disease diagnosis. As no requirement of any further functionalization of these as-prepared nanomaterials, this sensing system shows remarkable advantages including very fast and simple, cost-effective as well as environmental-friendly, which suggest that this new strategy could serve as an efficient tool for analyzing GSH level in biosamples.
Keywords: Carbon dots; MnO2 nanosheets; Nanocomposites; Fluorescnece detection; Glutathione.
1. Introduction Glutathione (GSH), a tripeptide, is produced endogenously from L-cysteine, glycine, and L-glutamic acid (Anderson ME. 1998). GSH plays key roles in biological systems and serves many cellular functions such as the maintenance of intracellular redox states, detoxification, and metabolism (Zhang et al., 2004; Kanzok et al., 2000; Lu SC., 1999; Krauth-Siegel et al., 2005). Meanwhile, abnormal levels of GSH are also associated with numerous clinical diseases including human immunodeficiency virus (HIV) (Samiec et al., 1998), Parkinson (Townsend et al., 2003), liver damage (Lu et al., 2009), diabetes (Herzenberg et al., 1997), Alzheimer (Pocernich et al., 2012), inflammatory (Rahman et al., 2004), and cardiovascular diseases (CVDs) (Refsum et al., 1998). Therefore, sensitive and selective techniques for GSH detection are critically important. As is well known, a host of various assays for GSH detection have been reported in the past decade. The more established strategies include surface enhanced Raman scattering (SERS) (Huang et al., 2009; Saha et al., 2013), electrochemical analysis (Calvo-Marzal et al., 2004; Ricci et al., 2006; Harfield et al., 2012), high performance liquid chromatography (HPLC) (Reed et al., 1980; Patterson et al., 2008), capillary electrophoresis (Kubalczyk et al., 2009), enzyme linked immunosorbent assay (ELISA) (Wawegama et al., 2014), etc. Although these conventional strategies exhibit
promising results for GSH detection, there are still some hindrances including time-consuming process, the utilization of sophisticated and specialized equipment, and requirement for skilled technicians. Comparably, fluorescence spectrometry has been emerging as a promising tool for GSH detection due to its advantages such as high sensitivity, good flexibility as well as apparent simplicity (Shao et al., 2010; Niu et al., 2012). Among these fluorescence methods, organic dyes have been widely used as fluorescent probes. To date, these fluorescence assays are mainly based on organic dyes (Xu and Hepel, 2011). Unfortunately, most of organic dyes have poor photostability, narrow excitation spectra, and broad emission bands with red tailing, which have intrinsically confined their further applications. Recently, fluorescent nanomaterials have attracted tons of attentions for GSH detection such as Quantum dots (QDs) (Banerjee et al., 2009; Han et al., 2009; Liu et al., 2010), silver and gold nanoparticles (Shen et al., 2014; García-Marín et al., 2014; Zhu et al., 2014), upconversion nanoparticles (UCNPs) (Deng et al., 2011), and graphitic-C3N4 (Zhang et al., 2014). Wang’s group (Han et al., 2009) proposed a sensitive fluorescent sensor for biothiols detection based on the recovered fluorescence of CdTe quantum dots-Hg(II) system. However, the toxic heavy metal Cd2+ and Hg2+ ions posed a big threat to environment. Furthermore, Ag and Au nanoparticles-based procedure for GSH detection also needs the attendance of toxic Hg2+ ions. UCNPs, a class of rare-earth metal co-doped nanocrystals, own many attractive properties such as weak autofluorescence backgrounds and low toxicity. However, UCNPs also suffer some limitations such as complicated synthesis, high cost and overheating effect from a relatively high power density (102 - 103 mW cm-1) NIR laser. Recently, Zhang et al (Zhang et al., 2014) established a sensing system for GSH analysis by using graphitic-C3N4, which is a novel fluorescence carbon nanomaterial. Nevertheless, synthesis of graphitic-C3N4 nanosheet requires harsh terms such as high temperature of 550 ℃, strong acid treatment, etc. Also, study of graphitic-C3N4 is still remaining at a very early stage, whose quantum yield and dispersibility needs to be further improved. Consequently, a novel fluorescent nanomaterial with ideal bio- and environmental safety, high fluorescent intensity, simple operability, and low-cost property is highly desirable for
GSH analysis with low concentrations. Carbon dots (C-dots), a new class of fluorescent nanoparticles, have drawn increasing attention in recent years due to their exceptional advantages such as highly stable fluorescence intensity, tunable excitation and emission spectra, good resistance to photo- and chemical degradation, low toxicity, and excellent biocompatibility (Ding et al., 2013; Zhu et al., 2013; Qian et al., 2014). Meanwhile, compared with other fluorescent carbon nanomaterials such as graphene (Wang et al., 2014) and g-C3N4 (Zhang et al., 2014), C-dots have much more sources of raw materials, rather high quantum yield, and very good dispersibility in aqueous. These unique characteristics have spurned intense interests in the use of C-dots for bioassays and biosensors, which indicate that C-dots might be the most promising for quantifying trace amount of GSH. To the best of our knowledge, the investigations of C-dots-based GSH assay are remaining at a very early stage. Recently, Shi et al proposed a nanosensor based on C-dots and Au nanoparticles for sensitive GSH detection (Shi et al., 2014). However, this procedure needs to incubate GSH with Au nanoparticles for a while first and then another incubation time with C-dots, which make it rather complicated and limited its further applications in one-step real time analysis. In this study, we report for the first time a facile one-step approach for rapid and selective sensing of GSH by using C-dots–MnO2 nanocomposites. The principle of this strategy was demonstrated in Scheme 1. C-dots-MnO2 nanocomposites were fabricated through in situ synthesis of MnO2 nanosheets in C-dots colloidal solutions. As soon as C-dots–MnO2 nanocomposites were formed, the fluorescence of C-dots could be quenched thoroughly because the distance between C-dots and MnO2 nanosheets is close enough and consequently FRET happened. In the presence of GSH, MnO2 nanosheets would be reduced to Mn2+ ions. As a result, C-dots were released and the fluorescence was recovered subsequently with the enhancement directly related to GSH concentration. This proposed assay is simple, very fast, one-step, environmental friendly, and highly selective toward GSH. Under optimal conditions, a detection limit of 300 nM for GSH can be realized in aqueous solution. Furthermore, this strategy can also be applied for measuring GSH in human serum samples.
Preferred position for (Scheme 1)
2. Experimental 2.1 Reagents and Apparatus GSH (reduced form), Citric acid, ethanediamine, 2-(N-morpholino) ethanesulfonic acid (MES), and bovine serum albumin (BSA) were obtained from J&K Scientific Ltd (Beiijing, China). L-Glycine (Gly), L-leucine (Leu), L-isoleucine (Ile), L-lysine (Lys), L-proline (Pro), L-serine (Ser), L-valine (Val), L-tyrosine (Tyr), glucose, and tri(hydroxymethyl)aminomethane were purchased from Aladdin reagent Co., Ltd (Shanghai, China). All other reagents of analytical grade were purchased from J&K Scientific Ltd (Beiijing, China). All reagents were used as received without further purification. All solutions were prepared using ultrapure water generated by a Millipore Milli-Q water purification system (Billerica, MA, USA) with an electric resistance ≥18.2 MΩ. UV-vis measurements were carried out on a TU-1810 spectrophotometer (Beijing, China) and the spectra were collected from 250 nm to 800 nm. Fluorescence measurements were carried out on an F-4600 spectrophotometer (Hitachi, Japan). Fluorescence emission spectra were recorded from 385 nm to 620 nm at room temperature with an excitation wavelength at 360 nm. Fourier-transform infrared spectrum (FT-IR) was collected in the range of 4000-400 cm-1 by using a BRUKER TENSOR 27 spectrophotometer (Bruker, Germany) with KBr pellets. Morphology images of C-dots, MnO2 nanosheets, and C-dots-MnO2 nanocomposites were photographed by transmission electron microscope (FEI-Tecnai G2, USA) at an accelerating voltage of 200 kV. A drop of sample solution was placed on a copper grid that was left to dry before being transferred into the TEM sample chamber. 2.2 Preparation of Carbon Dots C-dots were synthesized by hydrothermal method according to the reported literature by using citric acid as carbon source (Zhu et al., 2013). Briefly, 1 g citric acid and 315 μL of ethanediamine were added into 10 mL of ultrapure water followed by
being stirred uniformly. The prepared solution was transferred into a stainless steel autoclave and heated at 300 ℃ for 5 h. After being cooled down to room temperature, the obtained dark brown product was centrifuged at 10000 rpm for 10 min to remove any precipitations, followed by being dialyzed against water with a cellulose ester dialysis membrane bag (molecular weight cutoff (MWCO) = 1000) for 24 hours. 2.3 Preparation of C-dots-MnO2 Nanocomposites In a typical reaction, 300 μL of C-dots was pipetted into a centrifuge tube containing 1.25 mL of 2-(N-morpholino) ethanesulfonic acid (MES) buffer (0.1 M, pH 6.0). Five hundred μL of KMnO4 (10 mM) solution was added to the above-mentioned tube. The final volume was adjusted to 5 mL by refilling ultrapure water. Then, the mixture was sonicated for 30 min until brown colloid solution formed. Subsequently, the obtained C-dots-MnO2 nanocomposites were collected by centrifugation at 10000 rpm for 5min and washed three times with ultrapure water. The as-prepared C-dots-MnO2 nanocomposites were redispersed in ultrapure water for the following studies. As a control, bare MnO2 nanosheets were prepared according to the above identical manner in the absence of C-dots. 2.4 Procedures for GSH Detecion The fluorescence detection of GSH was performed in ultrapure water. Different concentrations of GSH (0-2.5 mM) were mixed with 30 μL of the above-mentioned nanocomposites in a series of 500 μL centrifuge tubes. The mixture was diluted to 200 μL with ultrapure water, and then they were incubated at room temperature for 3 min. The fluorescence signal of nanocomposites was collected on a Hitachi F-4600 fluorescence spectrophotometer with excitation at 365 nm. For the kinetic study, the fluorescence recovery of nanocomposites was monitored in different incubation time by adding GSH (100 μM). 2.5 Specificity Investigation For the selectivity study, an aliquot of the stock solutions of nontarget samples (Gly, Leu, Ile, Lys, Pro, Tyr, Val, Ser) (10 mM), electrolytes (KCl, NaCl, MgCl2, MnCl2, NaSO4, MgSO4, PBS, Tris-HCl) (10 mM), BSA (10mg/mL) were prepared in ultrapure water. Twenty μL of interfering substances were mixed with 30 μL of the
above-mentioned nanocomposites which were adjusted to 200 μL with ultrapure water. Fluorescence spectra were recorded with excitation at 365 nm after the interferents were added to reaction solution for 3 min. 2.6 Sensing of GSH in Human Serum samples Human serum samples were treated by centrifugation at 10000 rpm for 10 min. The supernatant was mixed with GSH (30, 40 and 50 μM) in 500 μL centrifuge tube. Then, solution was measured after incubation at room temperature for 3 min. Finally, fluorescence intensities at a wavelength of 435 nm were recorded. Spectrofluorimetry experiments using molecular fluorescence probe o-phthalaldehyde (OPA) were also conducted to measure the GSH content in human serum sample as a reference method (Senft et al., 2000). 3. Results and Discussion 3.1 Synthesis and Characterization of C-dots, MnO2 Nanosheets, and C-dots-MnO2 Nanocomposites Citric acid and ethanediamine serving as raw materials were used to synthesize C-dots according to a previously reported method with minor modification (Zhu et al., 2013). UV-vis absorption and fluorescence spectra were recorded to investigate the optical properties of as-prepared C-dots. These C-dots aqueous solution has a UV absorption peak at 340 nm and exhibits strong blue emission at 435 nm under the excitation of 360 nm (Fig. 1A). The inset in Fig. 1A shows the photographs of C-dots solution under white light and a 365-nm UV lamp. Meanwhile, these C-dots are highly water soluble due to the existence of carboxyl and amino groups on their surface, which can be proved from the Fourier transform infrared (FT-IR) spectrum (Fig. S1). Accordingly, the peak at 3420 cm-1 and 3260 cm-1 are attributed to O-H and N-H stretching vibrations. Asymmetric stretching vibrations of =CH2, C-H, C=O, and N-H are at 3080 cm-1, 2964 cm-1, 1656 cm-1, and 1562 cm-1, respectively. Moreover, The as-synthesized C-dots are very stable for several month at room temperature without any precipitations, which facilitates their further applications.
C-dots-MnO2 nanocomposites were fabricated through in situ synthesis of MnO2 nanosheets in carbon dots colloid solution. In the absence of C-dots, only bare MnO2 nanosheets were generated. As can be seen from Fig. 1B, the bare MnO2 nanosheets have a broad UV-vis absorption spectrum from 250 nm to 500 nm, which is the same as the reported optical properties of MnO2 nanomaterials. More importantly, the absorption spectrum of MnO2 nanosheets has a large overlap with the fluorescence emission spectrum of C-dots, which results in the efficient FRET between C-dots and MnO2 nanosheets (Fig. 1B). Compared with the bared MnO2 nanosheets, C-dots-MnO2 nanocomposites have shown similar UV-vis spectrum whereas with a little blue shift, which might be due to the adherence of C-dots onto the surface of MnO2 nanosheets (Fig. 1C). In the presence of C-dots, C-dots-MnO2 nanocomposites could be fabricated in situ because C-dots would adhere to MnO2 nanosheets very easily as soon as these nanosheets formed. To further prove our products, morphology of C-dots, MnO2 nanosheets, and C-dots-MnO2 nanocomposites were characterized by TEM, respectively. Fig. 2A and 2B showed that both C-dots and MnO2 nanosheets were highly monodisperse and C-dots have uniform and small size less than 5 nm. Meanwhile, as shown in Fig. 2C, C-dots were successfully adhered to the surface of MnO2 nanosheets dominated by electrostatic interactions. Furthermore, the stability of C-dots-MnO2 nanocomposites has been investigated and the result showed that the fluorescence signal has little change even after the nanocomposites were stored at RT for 10 days (Fig. S3).
3.2 Optimization of Experimental Conditions Obviously, the concentration of KMnO4 has important influences on the fluorescence of C-dots since more KMnO4 will generate more MnO2 nanosheets. As shown in Fig. S2, the fluorescence intensity of C-dots decreased gradually along with the increasing of KMnO4 concentrations. When the concentration of KMnO4 was higher than 1 mM, the quenching efficiency was slow down and then reached equilibrium. Finally, 1 mM KMnO4 was chosen as the optimal amount for the subsequent experiments. To determine optimal dosage of nanocomposites, fluorescence recovery of
different volumes of nanocomposites after adding 0.1 mM GSH was recorded for detection effect study. As can be concluded from Fig. S5, 30 μL of nanocomposites was selected as the best candidate. Another parameter as assay time was also optimized as shown in Fig. S6. When GSH was added into C-dots-MnO2 solution, the fluorescence signal increased rapidly with the increasing of incubation time and then reached to a stable stage only in 3 min. Thus, 3 min was chosen as the appropriate time in the following experiments, which is much faster compared with the reported assays. 3.3 Detection of GSH To demonstrate the applicability of this developed fluorescence “switch-on” sensor for GSH detection, we tested the response of this assay to different concentrations of GSH under optimal conditions. As shown in Fig. 3A, the fluorescence signal of C-dots was gradually restored as the concentration of GSH varied from 0 to 250 μM. The restored fluorescence was dependent on the amount of GSH. When the concentration of GSH was increased to 250 μM, no further restoring of fluorescence can be observed, showing that the sensing response has reached the maximum. Meanwhile, a good linear relationship over the range from 1 to 10 μM with a correlation coefficient square of 0.9967 was obtained (Fig. 3B, F and F0 are fluorescence intensities in the presence and absence of GSH, respectively). The detection limit of this assay for GSH was 300 nM according to the 3σ rule, which indicated that this proposed sensing procedure could satisfy the clinical and medicinal requirements about sensitive GSH detection. In addition, we further compared the performance of this approach with other reported methods for GSH determination, which was shown in Table S1. The result indicated that this proposed procedure had good sensitivity and also exhibited much shorter assay time than many previous methods.
3.4 Selectivity of C-dots-MnO2 Nanocomposites-based GSH assay To evaluate the selectivity of this assay, we investigated its fluorescence response towards some electrolytes, amino acids, and proteins. As shown in Fig. 4, the fluorescence intensities in the presence of 10 μM and 100 μM GSH were strikingly
larger than that of other amino acids, electrotypes and proteins with relative higher concentrations. This result revealed that a wide range of electrolytes and weakly reducing bioagents such as glucose did not lead to notable optical responses, which indicates that this proposed assay is highly selective toward GSH over other nontarget samples. Although cysteine (Cys) and homocysteine (HCys) also can cause fluorescence response to this system, whose content (μM levels) is much lower than that of GSH (mM levels) in biological systems (Yu et al., 2013; Saha and Jana, 2013). Thus, the C-dots–MnO2 nanocomposites could be applied as a selective nanosensor for GSH detection without any significant interference. In addition, as different methods for GSH detection were compared with this work.
3.5 Determination of GSH in Human Serum To explore the feasibility of this proposed procedure in complicated biological environment, the assay was then applied to detect GSH spiked in diluted human serum. Due to high concentration of GSH in human serum (mM levels), the human serum was diluted so that original GSH concentration could fall in the standard calibration curve of this assay, which also showed that our assay has sufficient sensitivity and only require small amount of real samples. After that, 3.0 μM, 4.0 μM, and 5.0 μM GSH were spiked into the diluted serum samples, respectively, and then was added into the C-dots-MnO2 solution. The fluorescence signal was recorded by the F-4600 spectrophotometer after 3 min. All the data are based on three duplicated measurements and shown in Table 1. According to the calibration curve obtained in aqueous solution (Fig. 3B), the original GSH concentration in the human serum sample was determined as 1.304 μM, which is consistent with the result when using OPA probe (1.325 μM). Furthermore, recoveries of the known spiked amounts of GSH in human serum were obtained in the range of 94.6%–105.1% (Table 1) and a good linear relationship was realized in the dosing experiment for human serum samples (Fig. S7), indicating the good reliability of this strategy. Meanwhile, we further tested 5 human serum samples by using this method and validated the results with that of OPA probe (Table S2). Apparently, successfully
detecting GSH in human serum displays the promise of C-dots-MnO2 nanocomposite for GSH measuring even in complicated biological environments.
4. Conclusion In this work, we developed a new fluorescence “switch-on” assay for GSH detection based on the in situ synthesized C-dots-MnO2 nanocomposite. This novel proposed method offers strong detection ability for GSH with a wide concentration range as well as a detection limit of 300 nM. Meanwhile, this assay could not only function in aqueous solution for GSH detection but also exhibit reliable responses toward GSH in human serum samples. Taking full advantages of C-dots-MnO2 nanocomposite, this assay shows remarkable properties including very fast and simple, one-step, cost-effective as well as environmental-friendly, which suggest that this new strategy has a great potential to be utilized as an efficient tool for analyzing GSH level in biological environments.
Acknowledgments This work was supported by the National Natural Science Foundation of China (21205108), the Scientific Research Foundation for the Returned Overseas Chinese Scholars (State Education Ministry of China), and the Technology Foundation for Selected Overseas Chinese Scholars (Ministry of Personnel of China).
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Figure captions Scheme 1. Schematic principle of C-dots-MnO2 nanocomposites for GSH detection.
Fig. 1. (A) UV-vis absorption and fluorescence spectra of C-dots in aqueous solution. Inset shows the color change of C-dots solution without and with UV irradiation. (B) Spectral overlap showing the UV-vis absorption spectrum of MnO2 nanosheets (black)
and the fluorescence emission spectrum of C-dots (blue). (C) UV-Vis absorption spectra of KMnO4 (purple), MnO2 nanosheets (black), and C-dots-MnO2 nanocomposites (red) in aqueous solution. Fig. 2. TEM images of C-dots (A), MnO2 nanosheets (B), and C-dots-MnO2 nanocomposites (C).
Fig. 3. (A) Fluorescence recovery spectra of carbon dots quenched with MnO2 nanosheets by addition of GSH with concentration ranging from 0 to 250 μM (Inset: Plot of relative fluorescence intensity against GSH concentration); (B) Linear relationship between relative fluorescence intensity and GSH concentration. F0 represents the fluorescence intensity without GSH, and F represents the fluorescence intensity with different GSH concentration (λex=360 nm; λem=435 nm).
Fig. 4. Relative fluorescence intensity of C-Dots-MnO2 nanocomposites in the presence of various biomolecules and electrolytes (1mM each; BSA: 1mg/mL; PBS: 10mM, pH=7.4; Tris-HCl: 10mM, pH=7.4), where F0 represents the fluorescence intensity without GSH or nontarget samples, and F represents the fluorescence intensity with different concentration of GSH or nontarget samples (λex=360 nm; λem=435 nm).
Table 1. Determination of GSH in human serum
Method
This method
OPA probe
Found in
Added
Total found
Recovery
RSD
sample/μM
/μM
/μM
/%, n=3
/%, n=3
1.304
3.0
4.141
94.6
2.9
4.0
5.279
99.4
3.4
5.0
6.557
105.1
3.7
3.0
4.198
95.8
2.5
4.0
5.455
103.3
3.1
5.0
6.235
98.2
3.3
1.325
Highlights
A novel fluorescence “switch-on” biosensor is designed for glutathione detection by using carbon dots-MnO2 nanocomposites. This sensing system displays remarkable properties including simplicity, very short assay time, low cost as well as nontoxicity. Results for human serum samples demonstrated that this biosensor is highly applicable for the detection of GSH in biological environments.
PII: DOI: Reference:
S0956-5663(15)30077-4 http://dx.doi.org/10.1016/j.bios.2015.04.077 BIOS7645
To appear in: Biosensors and Bioelectronic Received date: 3 February 2015 Revised date: 12 April 2015 Accepted date: 23 April 2015 Cite this article as: Qi-Yong Cai, Jie Li, Jia Ge, Lin Zhang, Ya-Lei Hu, ZhaoHui Li and Ling-Bo Qu, A rapid fluorescence “switch-on” assay for Glutathione detection by using carbon Dots-MnO2 nanocomposites, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.04.077 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A Rapid Fluorescence “Switch-On” Assay for Glutathione Detection by using Carbon Dots-MnO2 Nanocomposites Qi-Yong Caia,1, Jie Lia,1, Jia Gea,b, Lin Zhanga, Ya-Lei Hua, Zhao-Hui Lia,b,*, and Ling-Bo Qua,c,*
a
College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou
450001, P. R. China b
State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University,
Changsha 410082, P.R. China c
School of Chemistry & Chemical Engineering, Henan University of Technology, Zhengzhou 450001, P.R. China
*
Corresponding authors. Tel.: +86-371-67783126; Fax: +86-371-67781556.
E-mail address: [email protected] (Z. H. Li), [email protected] (L. B. Qu). 1 These authors contributed equally to this work. Abstract Glutathione (GSH) serves many cellular functions and plays crucial roles in human pathologies. Simple and sensitive sensors capable of detecting GSH would be useful tools to understand the mechanism of diseases. In this work, a rapid fluorescence “switch-on” assay was developed to detect trace amount of GSH based on carbon dots-MnO2 nanocomposites, which was fabricated through in situ synthesis of MnO2 nanosheets in carbon dots colloid solution. Due to the formation of carbon dots-MnO2 nanocomposites, fluorescence of carbon dots could be quenched efficiently by MnO2 nanosheeets through fluorescence resonance energy transfer (FRET). However, the presence of GSH would reduce MnO2 nanosheets to Mn2+ ions and subsequently release
carbon dots, which resulted in sufficient recovery of fluorescent signal. This proposed assay demonstrated highly selectivity toward GSH with a detection limit of 300 nM. Moreover, this method has also shown sensitive responses to GSH in human serum samples, which indicated its great potential to be used in disease diagnosis. As no requirement of any further functionalization of these as-prepared nanomaterials, this sensing system shows remarkable advantages including very fast and simple, cost-effective as well as environmental-friendly, which suggest that this new strategy could serve as an efficient tool for analyzing GSH level in biosamples.
Keywords: Carbon dots; MnO2 nanosheets; Nanocomposites; Fluorescnece detection; Glutathione.
1. Introduction Glutathione (GSH), a tripeptide, is produced endogenously from L-cysteine, glycine, and L-glutamic acid (Anderson ME. 1998). GSH plays key roles in biological systems and serves many cellular functions such as the maintenance of intracellular redox states, detoxification, and metabolism (Zhang et al., 2004; Kanzok et al., 2000; Lu SC., 1999; Krauth-Siegel et al., 2005). Meanwhile, abnormal levels of GSH are also associated with numerous clinical diseases including human immunodeficiency virus (HIV) (Samiec et al., 1998), Parkinson (Townsend et al., 2003), liver damage (Lu et al., 2009), diabetes (Herzenberg et al., 1997), Alzheimer (Pocernich et al., 2012), inflammatory (Rahman et al., 2004), and cardiovascular diseases (CVDs) (Refsum et al., 1998). Therefore, sensitive and selective techniques for GSH detection are critically important. As is well known, a host of various assays for GSH detection have been reported in the past decade. The more established strategies include surface enhanced Raman scattering (SERS) (Huang et al., 2009; Saha et al., 2013), electrochemical analysis (Calvo-Marzal et al., 2004; Ricci et al., 2006; Harfield et al., 2012), high performance liquid chromatography (HPLC) (Reed et al., 1980; Patterson et al., 2008), capillary electrophoresis (Kubalczyk et al., 2009), enzyme linked immunosorbent assay (ELISA) (Wawegama et al., 2014), etc. Although these conventional strategies exhibit
promising results for GSH detection, there are still some hindrances including time-consuming process, the utilization of sophisticated and specialized equipment, and requirement for skilled technicians. Comparably, fluorescence spectrometry has been emerging as a promising tool for GSH detection due to its advantages such as high sensitivity, good flexibility as well as apparent simplicity (Shao et al., 2010; Niu et al., 2012). Among these fluorescence methods, organic dyes have been widely used as fluorescent probes. To date, these fluorescence assays are mainly based on organic dyes (Xu and Hepel, 2011). Unfortunately, most of organic dyes have poor photostability, narrow excitation spectra, and broad emission bands with red tailing, which have intrinsically confined their further applications. Recently, fluorescent nanomaterials have attracted tons of attentions for GSH detection such as Quantum dots (QDs) (Banerjee et al., 2009; Han et al., 2009; Liu et al., 2010), silver and gold nanoparticles (Shen et al., 2014; García-Marín et al., 2014; Zhu et al., 2014), upconversion nanoparticles (UCNPs) (Deng et al., 2011), and graphitic-C3N4 (Zhang et al., 2014). Wang’s group (Han et al., 2009) proposed a sensitive fluorescent sensor for biothiols detection based on the recovered fluorescence of CdTe quantum dots-Hg(II) system. However, the toxic heavy metal Cd2+ and Hg2+ ions posed a big threat to environment. Furthermore, Ag and Au nanoparticles-based procedure for GSH detection also needs the attendance of toxic Hg2+ ions. UCNPs, a class of rare-earth metal co-doped nanocrystals, own many attractive properties such as weak autofluorescence backgrounds and low toxicity. However, UCNPs also suffer some limitations such as complicated synthesis, high cost and overheating effect from a relatively high power density (102 - 103 mW cm-1) NIR laser. Recently, Zhang et al (Zhang et al., 2014) established a sensing system for GSH analysis by using graphitic-C3N4, which is a novel fluorescence carbon nanomaterial. Nevertheless, synthesis of graphitic-C3N4 nanosheet requires harsh terms such as high temperature of 550 ℃, strong acid treatment, etc. Also, study of graphitic-C3N4 is still remaining at a very early stage, whose quantum yield and dispersibility needs to be further improved. Consequently, a novel fluorescent nanomaterial with ideal bio- and environmental safety, high fluorescent intensity, simple operability, and low-cost property is highly desirable for
GSH analysis with low concentrations. Carbon dots (C-dots), a new class of fluorescent nanoparticles, have drawn increasing attention in recent years due to their exceptional advantages such as highly stable fluorescence intensity, tunable excitation and emission spectra, good resistance to photo- and chemical degradation, low toxicity, and excellent biocompatibility (Ding et al., 2013; Zhu et al., 2013; Qian et al., 2014). Meanwhile, compared with other fluorescent carbon nanomaterials such as graphene (Wang et al., 2014) and g-C3N4 (Zhang et al., 2014), C-dots have much more sources of raw materials, rather high quantum yield, and very good dispersibility in aqueous. These unique characteristics have spurned intense interests in the use of C-dots for bioassays and biosensors, which indicate that C-dots might be the most promising for quantifying trace amount of GSH. To the best of our knowledge, the investigations of C-dots-based GSH assay are remaining at a very early stage. Recently, Shi et al proposed a nanosensor based on C-dots and Au nanoparticles for sensitive GSH detection (Shi et al., 2014). However, this procedure needs to incubate GSH with Au nanoparticles for a while first and then another incubation time with C-dots, which make it rather complicated and limited its further applications in one-step real time analysis. In this study, we report for the first time a facile one-step approach for rapid and selective sensing of GSH by using C-dots–MnO2 nanocomposites. The principle of this strategy was demonstrated in Scheme 1. C-dots-MnO2 nanocomposites were fabricated through in situ synthesis of MnO2 nanosheets in C-dots colloidal solutions. As soon as C-dots–MnO2 nanocomposites were formed, the fluorescence of C-dots could be quenched thoroughly because the distance between C-dots and MnO2 nanosheets is close enough and consequently FRET happened. In the presence of GSH, MnO2 nanosheets would be reduced to Mn2+ ions. As a result, C-dots were released and the fluorescence was recovered subsequently with the enhancement directly related to GSH concentration. This proposed assay is simple, very fast, one-step, environmental friendly, and highly selective toward GSH. Under optimal conditions, a detection limit of 300 nM for GSH can be realized in aqueous solution. Furthermore, this strategy can also be applied for measuring GSH in human serum samples.
Preferred position for (Scheme 1)
2. Experimental 2.1 Reagents and Apparatus GSH (reduced form), Citric acid, ethanediamine, 2-(N-morpholino) ethanesulfonic acid (MES), and bovine serum albumin (BSA) were obtained from J&K Scientific Ltd (Beiijing, China). L-Glycine (Gly), L-leucine (Leu), L-isoleucine (Ile), L-lysine (Lys), L-proline (Pro), L-serine (Ser), L-valine (Val), L-tyrosine (Tyr), glucose, and tri(hydroxymethyl)aminomethane were purchased from Aladdin reagent Co., Ltd (Shanghai, China). All other reagents of analytical grade were purchased from J&K Scientific Ltd (Beiijing, China). All reagents were used as received without further purification. All solutions were prepared using ultrapure water generated by a Millipore Milli-Q water purification system (Billerica, MA, USA) with an electric resistance ≥18.2 MΩ. UV-vis measurements were carried out on a TU-1810 spectrophotometer (Beijing, China) and the spectra were collected from 250 nm to 800 nm. Fluorescence measurements were carried out on an F-4600 spectrophotometer (Hitachi, Japan). Fluorescence emission spectra were recorded from 385 nm to 620 nm at room temperature with an excitation wavelength at 360 nm. Fourier-transform infrared spectrum (FT-IR) was collected in the range of 4000-400 cm-1 by using a BRUKER TENSOR 27 spectrophotometer (Bruker, Germany) with KBr pellets. Morphology images of C-dots, MnO2 nanosheets, and C-dots-MnO2 nanocomposites were photographed by transmission electron microscope (FEI-Tecnai G2, USA) at an accelerating voltage of 200 kV. A drop of sample solution was placed on a copper grid that was left to dry before being transferred into the TEM sample chamber. 2.2 Preparation of Carbon Dots C-dots were synthesized by hydrothermal method according to the reported literature by using citric acid as carbon source (Zhu et al., 2013). Briefly, 1 g citric acid and 315 μL of ethanediamine were added into 10 mL of ultrapure water followed by
being stirred uniformly. The prepared solution was transferred into a stainless steel autoclave and heated at 300 ℃ for 5 h. After being cooled down to room temperature, the obtained dark brown product was centrifuged at 10000 rpm for 10 min to remove any precipitations, followed by being dialyzed against water with a cellulose ester dialysis membrane bag (molecular weight cutoff (MWCO) = 1000) for 24 hours. 2.3 Preparation of C-dots-MnO2 Nanocomposites In a typical reaction, 300 μL of C-dots was pipetted into a centrifuge tube containing 1.25 mL of 2-(N-morpholino) ethanesulfonic acid (MES) buffer (0.1 M, pH 6.0). Five hundred μL of KMnO4 (10 mM) solution was added to the above-mentioned tube. The final volume was adjusted to 5 mL by refilling ultrapure water. Then, the mixture was sonicated for 30 min until brown colloid solution formed. Subsequently, the obtained C-dots-MnO2 nanocomposites were collected by centrifugation at 10000 rpm for 5min and washed three times with ultrapure water. The as-prepared C-dots-MnO2 nanocomposites were redispersed in ultrapure water for the following studies. As a control, bare MnO2 nanosheets were prepared according to the above identical manner in the absence of C-dots. 2.4 Procedures for GSH Detecion The fluorescence detection of GSH was performed in ultrapure water. Different concentrations of GSH (0-2.5 mM) were mixed with 30 μL of the above-mentioned nanocomposites in a series of 500 μL centrifuge tubes. The mixture was diluted to 200 μL with ultrapure water, and then they were incubated at room temperature for 3 min. The fluorescence signal of nanocomposites was collected on a Hitachi F-4600 fluorescence spectrophotometer with excitation at 365 nm. For the kinetic study, the fluorescence recovery of nanocomposites was monitored in different incubation time by adding GSH (100 μM). 2.5 Specificity Investigation For the selectivity study, an aliquot of the stock solutions of nontarget samples (Gly, Leu, Ile, Lys, Pro, Tyr, Val, Ser) (10 mM), electrolytes (KCl, NaCl, MgCl2, MnCl2, NaSO4, MgSO4, PBS, Tris-HCl) (10 mM), BSA (10mg/mL) were prepared in ultrapure water. Twenty μL of interfering substances were mixed with 30 μL of the
above-mentioned nanocomposites which were adjusted to 200 μL with ultrapure water. Fluorescence spectra were recorded with excitation at 365 nm after the interferents were added to reaction solution for 3 min. 2.6 Sensing of GSH in Human Serum samples Human serum samples were treated by centrifugation at 10000 rpm for 10 min. The supernatant was mixed with GSH (30, 40 and 50 μM) in 500 μL centrifuge tube. Then, solution was measured after incubation at room temperature for 3 min. Finally, fluorescence intensities at a wavelength of 435 nm were recorded. Spectrofluorimetry experiments using molecular fluorescence probe o-phthalaldehyde (OPA) were also conducted to measure the GSH content in human serum sample as a reference method (Senft et al., 2000). 3. Results and Discussion 3.1 Synthesis and Characterization of C-dots, MnO2 Nanosheets, and C-dots-MnO2 Nanocomposites Citric acid and ethanediamine serving as raw materials were used to synthesize C-dots according to a previously reported method with minor modification (Zhu et al., 2013). UV-vis absorption and fluorescence spectra were recorded to investigate the optical properties of as-prepared C-dots. These C-dots aqueous solution has a UV absorption peak at 340 nm and exhibits strong blue emission at 435 nm under the excitation of 360 nm (Fig. 1A). The inset in Fig. 1A shows the photographs of C-dots solution under white light and a 365-nm UV lamp. Meanwhile, these C-dots are highly water soluble due to the existence of carboxyl and amino groups on their surface, which can be proved from the Fourier transform infrared (FT-IR) spectrum (Fig. S1). Accordingly, the peak at 3420 cm-1 and 3260 cm-1 are attributed to O-H and N-H stretching vibrations. Asymmetric stretching vibrations of =CH2, C-H, C=O, and N-H are at 3080 cm-1, 2964 cm-1, 1656 cm-1, and 1562 cm-1, respectively. Moreover, The as-synthesized C-dots are very stable for several month at room temperature without any precipitations, which facilitates their further applications.
C-dots-MnO2 nanocomposites were fabricated through in situ synthesis of MnO2 nanosheets in carbon dots colloid solution. In the absence of C-dots, only bare MnO2 nanosheets were generated. As can be seen from Fig. 1B, the bare MnO2 nanosheets have a broad UV-vis absorption spectrum from 250 nm to 500 nm, which is the same as the reported optical properties of MnO2 nanomaterials. More importantly, the absorption spectrum of MnO2 nanosheets has a large overlap with the fluorescence emission spectrum of C-dots, which results in the efficient FRET between C-dots and MnO2 nanosheets (Fig. 1B). Compared with the bared MnO2 nanosheets, C-dots-MnO2 nanocomposites have shown similar UV-vis spectrum whereas with a little blue shift, which might be due to the adherence of C-dots onto the surface of MnO2 nanosheets (Fig. 1C). In the presence of C-dots, C-dots-MnO2 nanocomposites could be fabricated in situ because C-dots would adhere to MnO2 nanosheets very easily as soon as these nanosheets formed. To further prove our products, morphology of C-dots, MnO2 nanosheets, and C-dots-MnO2 nanocomposites were characterized by TEM, respectively. Fig. 2A and 2B showed that both C-dots and MnO2 nanosheets were highly monodisperse and C-dots have uniform and small size less than 5 nm. Meanwhile, as shown in Fig. 2C, C-dots were successfully adhered to the surface of MnO2 nanosheets dominated by electrostatic interactions. Furthermore, the stability of C-dots-MnO2 nanocomposites has been investigated and the result showed that the fluorescence signal has little change even after the nanocomposites were stored at RT for 10 days (Fig. S3).
3.2 Optimization of Experimental Conditions Obviously, the concentration of KMnO4 has important influences on the fluorescence of C-dots since more KMnO4 will generate more MnO2 nanosheets. As shown in Fig. S2, the fluorescence intensity of C-dots decreased gradually along with the increasing of KMnO4 concentrations. When the concentration of KMnO4 was higher than 1 mM, the quenching efficiency was slow down and then reached equilibrium. Finally, 1 mM KMnO4 was chosen as the optimal amount for the subsequent experiments. To determine optimal dosage of nanocomposites, fluorescence recovery of
different volumes of nanocomposites after adding 0.1 mM GSH was recorded for detection effect study. As can be concluded from Fig. S5, 30 μL of nanocomposites was selected as the best candidate. Another parameter as assay time was also optimized as shown in Fig. S6. When GSH was added into C-dots-MnO2 solution, the fluorescence signal increased rapidly with the increasing of incubation time and then reached to a stable stage only in 3 min. Thus, 3 min was chosen as the appropriate time in the following experiments, which is much faster compared with the reported assays. 3.3 Detection of GSH To demonstrate the applicability of this developed fluorescence “switch-on” sensor for GSH detection, we tested the response of this assay to different concentrations of GSH under optimal conditions. As shown in Fig. 3A, the fluorescence signal of C-dots was gradually restored as the concentration of GSH varied from 0 to 250 μM. The restored fluorescence was dependent on the amount of GSH. When the concentration of GSH was increased to 250 μM, no further restoring of fluorescence can be observed, showing that the sensing response has reached the maximum. Meanwhile, a good linear relationship over the range from 1 to 10 μM with a correlation coefficient square of 0.9967 was obtained (Fig. 3B, F and F0 are fluorescence intensities in the presence and absence of GSH, respectively). The detection limit of this assay for GSH was 300 nM according to the 3σ rule, which indicated that this proposed sensing procedure could satisfy the clinical and medicinal requirements about sensitive GSH detection. In addition, we further compared the performance of this approach with other reported methods for GSH determination, which was shown in Table S1. The result indicated that this proposed procedure had good sensitivity and also exhibited much shorter assay time than many previous methods.
3.4 Selectivity of C-dots-MnO2 Nanocomposites-based GSH assay To evaluate the selectivity of this assay, we investigated its fluorescence response towards some electrolytes, amino acids, and proteins. As shown in Fig. 4, the fluorescence intensities in the presence of 10 μM and 100 μM GSH were strikingly
larger than that of other amino acids, electrotypes and proteins with relative higher concentrations. This result revealed that a wide range of electrolytes and weakly reducing bioagents such as glucose did not lead to notable optical responses, which indicates that this proposed assay is highly selective toward GSH over other nontarget samples. Although cysteine (Cys) and homocysteine (HCys) also can cause fluorescence response to this system, whose content (μM levels) is much lower than that of GSH (mM levels) in biological systems (Yu et al., 2013; Saha and Jana, 2013). Thus, the C-dots–MnO2 nanocomposites could be applied as a selective nanosensor for GSH detection without any significant interference. In addition, as different methods for GSH detection were compared with this work.
3.5 Determination of GSH in Human Serum To explore the feasibility of this proposed procedure in complicated biological environment, the assay was then applied to detect GSH spiked in diluted human serum. Due to high concentration of GSH in human serum (mM levels), the human serum was diluted so that original GSH concentration could fall in the standard calibration curve of this assay, which also showed that our assay has sufficient sensitivity and only require small amount of real samples. After that, 3.0 μM, 4.0 μM, and 5.0 μM GSH were spiked into the diluted serum samples, respectively, and then was added into the C-dots-MnO2 solution. The fluorescence signal was recorded by the F-4600 spectrophotometer after 3 min. All the data are based on three duplicated measurements and shown in Table 1. According to the calibration curve obtained in aqueous solution (Fig. 3B), the original GSH concentration in the human serum sample was determined as 1.304 μM, which is consistent with the result when using OPA probe (1.325 μM). Furthermore, recoveries of the known spiked amounts of GSH in human serum were obtained in the range of 94.6%–105.1% (Table 1) and a good linear relationship was realized in the dosing experiment for human serum samples (Fig. S7), indicating the good reliability of this strategy. Meanwhile, we further tested 5 human serum samples by using this method and validated the results with that of OPA probe (Table S2). Apparently, successfully
detecting GSH in human serum displays the promise of C-dots-MnO2 nanocomposite for GSH measuring even in complicated biological environments.
4. Conclusion In this work, we developed a new fluorescence “switch-on” assay for GSH detection based on the in situ synthesized C-dots-MnO2 nanocomposite. This novel proposed method offers strong detection ability for GSH with a wide concentration range as well as a detection limit of 300 nM. Meanwhile, this assay could not only function in aqueous solution for GSH detection but also exhibit reliable responses toward GSH in human serum samples. Taking full advantages of C-dots-MnO2 nanocomposite, this assay shows remarkable properties including very fast and simple, one-step, cost-effective as well as environmental-friendly, which suggest that this new strategy has a great potential to be utilized as an efficient tool for analyzing GSH level in biological environments.
Acknowledgments This work was supported by the National Natural Science Foundation of China (21205108), the Scientific Research Foundation for the Returned Overseas Chinese Scholars (State Education Ministry of China), and the Technology Foundation for Selected Overseas Chinese Scholars (Ministry of Personnel of China).
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Figure captions Scheme 1. Schematic principle of C-dots-MnO2 nanocomposites for GSH detection.
Fig. 1. (A) UV-vis absorption and fluorescence spectra of C-dots in aqueous solution. Inset shows the color change of C-dots solution without and with UV irradiation. (B) Spectral overlap showing the UV-vis absorption spectrum of MnO2 nanosheets (black)
and the fluorescence emission spectrum of C-dots (blue). (C) UV-Vis absorption spectra of KMnO4 (purple), MnO2 nanosheets (black), and C-dots-MnO2 nanocomposites (red) in aqueous solution. Fig. 2. TEM images of C-dots (A), MnO2 nanosheets (B), and C-dots-MnO2 nanocomposites (C).
Fig. 3. (A) Fluorescence recovery spectra of carbon dots quenched with MnO2 nanosheets by addition of GSH with concentration ranging from 0 to 250 μM (Inset: Plot of relative fluorescence intensity against GSH concentration); (B) Linear relationship between relative fluorescence intensity and GSH concentration. F0 represents the fluorescence intensity without GSH, and F represents the fluorescence intensity with different GSH concentration (λex=360 nm; λem=435 nm).
Fig. 4. Relative fluorescence intensity of C-Dots-MnO2 nanocomposites in the presence of various biomolecules and electrolytes (1mM each; BSA: 1mg/mL; PBS: 10mM, pH=7.4; Tris-HCl: 10mM, pH=7.4), where F0 represents the fluorescence intensity without GSH or nontarget samples, and F represents the fluorescence intensity with different concentration of GSH or nontarget samples (λex=360 nm; λem=435 nm).
Table 1. Determination of GSH in human serum
Method
This method
OPA probe
Found in
Added
Total found
Recovery
RSD
sample/μM
/μM
/μM
/%, n=3
/%, n=3
1.304
3.0
4.141
94.6
2.9
4.0
5.279
99.4
3.4
5.0
6.557
105.1
3.7
3.0
4.198
95.8
2.5
4.0
5.455
103.3
3.1
5.0
6.235
98.2
3.3
1.325
Highlights
A novel fluorescence “switch-on” biosensor is designed for glutathione detection by using carbon dots-MnO2 nanocomposites. This sensing system displays remarkable properties including simplicity, very short assay time, low cost as well as nontoxicity. Results for human serum samples demonstrated that this biosensor is highly applicable for the detection of GSH in biological environments.
