Introducing Ratiometric Fluorescence to MnO2 Nanosheet-Based

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Introducing ratiometric fluorescence to MnO2 nanosheet-based biosensing: a simple, label-free ratiometric fluorescent sensor programmed by cascade logic circuit for ultrasensitive GSH detection Daoqing Fan, Changshuai Shang, Wenling Gu, Erkang Wang, and Shaojun Dong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b07369 • Publication Date (Web): 11 Jul 2017 Downloaded from http://pubs.acs.org on July 11, 2017

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ACS Applied Materials & Interfaces

Introducing Ratiometric Fluorescence to MnO2 Nanosheetbased

Biosensing:

a

Simple,

Label-free

Ratiometric

Fluorescent Sensor Programmed by Cascade Logic Circuit for Ultrasensitive GSH Detection Daoqing Fan, †‡ Changshuai Shang, †‡ Wenling Gu, †‡ Erkang Wang, †‡ Shaojun Dong †‡* †

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of

Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, China. ‡

University of Chinese Academy of Sciences, Beijing, 100039, China.

*Corresponding Author E-mail: [email protected].

KEYWORDS： Glutathione; MnO2 nanosheet; Ratiometric fluorescent sensor; Cascade logic circuit; Ultrasensitive detection. 1 Environment ACS Paragon Plus


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ABSTRACT

Glutathione (GSH) plays crucial roles in various biological functions, and the level alterations of which have been linked to varieties of diseases. Herein, we for the first time expanded the application of oxidase-like property of MnO2 nanosheet (MnO2 NS) to fluorescent substrates of peroxidase. Different from previously reported fluorescent quenching phenomena, we found that MnO2 NS could not only largely quench the fluorescence of highly-fluorescent Scopoletin (SC) but also surprisingly enhance that of non-fluorescent Amplex Red (AR) via oxidation reaction. If MnO2 NS was premixed with GSH, it will be reduced to Mn2+ and lose the oxidase-like property, accompanied with subsequent increase of SC’s fluorescence and decrease of AR’s. Based on above mechanism, we construct the first MnO2 NS-based ratiometric fluorescent sensor for ultrasensitive and selective detection of GSH. Notably, this ratiometric sensor is programmed by the cascade logic circuit (an INHIBIT gate cascade with a 1 to 2 decoder). And a linear relationship between ratiometric fluorescent intensities of the two substrates and logarithmic values of GSH’s concentrations is obtained. The detection limit of GSH is as low as 6.7 nM, which is much lower than previous ratiometric fluorescent sensors, and also the lowest MnO2 NS-based fluorescent GSH sensor reported so far. Furthermore, this sensor is simple, label-free and low-cost, it also presents excellent applicability in human serum samples.

2 Environment ACS Paragon Plus

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INTRODUCTION As the most ubiquitous intracellular non-protein thiol, glutathione (GSH) plays crucial roles in multifarious biological functions, including the maintenance of intracellular redox activities, gene regulation, intracellular signal transduction and xenobiotic metabolism.1 The level changes of GSH have been linked to varieties of diseases, such as leucocyte loss, psoriasis, liver damage, Parkinson and so on.

2, 3

Scientists have made great efforts to construct different systems for

sensitive and selective detection of GSH in vitro or in human serum samples, such as fluorescent,4,

5

colorimetric, 6 electrochemical, 7 surface-enhanced Raman scattering ones 8 and

so on.9-18 Though great achievements have been made, most of above systems were operated on the basis of artificially-synthesized organic dyes, labelled fluorophores and nanomaterials that require long time synthesis, complicated/tedious labelling and purification procedures. These drawbacks resulted in sophisticated test steps, high costs, general selectivity and sensitivity during GSH’s detection. Owing to which, exploring simple, low-cost and label-free systems for highly sensitive and selective monitoring GSH is ever important. Recently, as a kind of novel, facile-synthesized 2D nanomaterial with well biocompatibility, 19 single-layer manganese dioxide nanosheet (MnO2 NS) guided various systems for GSH’s detection

as

a

result

of

the

unique

reaction

between

MnO2

and

GSH

(MnO2+2GSH+2H+=Mn2++GSSG+2H2O).20 These systems can be illustrated from two aspects. On one hand, most of luminescent systems were operated by utilizing the efficient fluorescent quenching abilities of MnO2 NS towards labelled fluorophores, 18 organic dyes, 21, 22

19

quantum dots

and upconversion nanoparticles.20 To a certain extent, this strategy has led scientists to form

a customary perspective that MnO2 NS can only quench

23, 24

but not enhance the fluorescence

of luminophores. On the other hand, several colorimetric systems for GSH were also fabricated



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by taking advantage of the oxidase-like property of MnO2 NS and colorimetric substrates of peroxidase (such as 3, 3′, 5, 5′-tetramethylbenzidine, TMB).

25-27

Though this strategy could

produce visual signals (oxidized TMB with blue color) that recognized by the naked eye, it presented poor sensitivity/stability compared with above luminescent ones. More significantly, all of the MnO2 NS-based systems for GSH constructed so far are single-signal responsive (turnon or turn-off). Compared with target-triggered dual-signal responsive detection systems (such as ratiometric fluorescent), 4,

28

the single-signal ones 29 are restricted by non-negligible background

interferences, general reliability/accuracy of test results and so on. Ratiometric fluorescence has attracted increasing attentions in sensing and bio-imaging lately. Since the interfering environment/background factors are largely minimized through recording the ratio of fluorescent intensities at two different wavelengths,

30

this technique could achieve

more accurate and effective detection compared with single-signal steady-state fluorescence, which has enlightened many ratiometric fluorescent sensors for various targets.28-31 Besides, molecular logic computing

32-47

programmed analyses

28, 29, 37, 41

have gained extensive

advancements in recent decades as the inherent intelligence/stringency of Boolean logic can be brought into target analysis and this strategy also presents promising prospects for intelligent diagnostics of diseases in the future. However, to the best of our knowledge, MnO2 NS-based ratiometric fluorescent sensors for GSH have not been reported, and ratiometric sensors for GSH reported till now were typically based on artificially-synthesized organic dyes

48- 50

that require

tedious synthesis/purification or relied on heavy/noble metal ions which might bring sample contaminations. Moreover, these reported ratiometric sensors were never integrated with molecular logic computing. Under above background, a MnO2 NS-based simple, label-free


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ratiometric fluorescent sensor under the program of cascade logic circuit for ultrasensitive and selective detection of GSH is in highly demand and remains further study. Herein, we for the first time expanded the application of oxidase-like property of MnO2 NS to fluorescent substrates of peroxidase (Scopoletin, SC; Amplex Red, AR).51 Different from previous fluorescent quenching systems, we found that MnO2 NS could not only largely quench the fluorescence of highly-fluorescent SC but also surprisingly and obviously enhance that of non-fluorescent AR via oxidation reaction. If MnO2 NS was premixed with GSH, it will be reduced to Mn2+ and lose the oxidase-like property, accompanied with subsequent increase of SC’s fluorescence and decrease of AR’s. According to above mechanism, we construct the first MnO2 NS-based ratiometric fluorescent sensor (Scheme 1) that programmed by the cascade logic circuit (an INHIBIT gate cascade with a 1 to 2 decoder) for ultrasensitive and selective detection of GSH. And a linear relationship between ratiometric fluorescent values of the two substrates and logarithmic concentrations of GSH is obtained. The detection limit of GSH is as low as 6.7 nM, which is much lower than previous ratiometric fluorescent sensors for GSH, and also the lowest MnO2 NS-based fluorescent GSH sensor reported so far. Moreover, this sensor is simple, label-free and low-cost, it also presents excellent applicability in human serum samples. RESULTS AND DISCUSSION Synthesis and characterization of MnO2 NS The fast and simple synthesis of MnO2 NS was performed according to previous reports, 19, 20 in which sonication-induced reduction of KMnO4 in MES buffer was applied. As shown in Figure 1A, B, the obtained brown suspension presented obvious morphology of nanosheet (transmission electron microscopy (TEM) image), accompanied with the unique UV absorbance



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band appeared around 380 nm.27 Corresponding atomic force microscope (AFM) image showed that the thickness of MnO2 NS is about 4 nm (Figure S1), fully indicated the ultrathin structure of the nanosheet. The X-ray photoelectron spectra (XPS) was further applied to identify the existence of MnO2 NS, Figure 1C. Two characteristic peaks located at 642.0 eV (Mn 2p3/2) and 653.7 eV (Mn 2p1/2)

27

fully indicated the formation of MnO2 NS. All the above phenomena

adequately proved the successful preparation of ultrathin MnO2 NS. Oxidation of fluorescent substrates by MnO2 NS Previous application of oxidase-like property of MnO2 NS was confined to colorimetric substrates. While, the fluorescent substrates of peroxidase are also ideal candidates in biosensing 51

and DNA computing.

32, 33

Among various substrates, Scopoletin (SC) and Amplex Red (AR)

are two fluorescent ones that possess inverse responses.

51

Solely SC exhibits high signal at 465

nm but after H2O2’s oxidation under the catalysis of peroxidase, producing almost nonfluorescent product (FI465, SC-ox). By contrast, solely AR is non-fluorescent but could generate greatly enhanced signal at 585 nm (FI585, AR-ox) after H2O2’s oxidation. Considering the oxidase-like property of MnO2 NS,

25-27

we anticipate that it might oxidize AR and SC without

the participation of H2O2. As shown in Figure 2A (a, b), in the absence of MnO2 NS, SC presented high FI465 signal. After the oxidation of MnO2 NS, a greatly decreased signal was obtained. AR sole presented negligible FI585 signal (Figure 2 A (c)), but after oxidized by MnO2 NS, surprisingly, we observed an obviously enhanced FI585 signal (about 100 times), Figure 2A (d), which was quite different from fluorescent quenching phenomena in previous MnO2 NSbased biosensors. Furthermore, to ensure the non-interference of two substrates in one homogeneous system, the simultaneous use of them was also explored. The 3D fluorescent columns of two substrates in the absence/presence of MnO2 NS (Figure 2B, S1/S2 for SC,


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A1/A2 for AR) were just identical to their separate use. All the above phenomena certificated the efficient oxidation ability of MnO2 NS towards SC and AR. Corresponding optimization experiments (concentrations of MnO2 NS, oxidation time of SC and AR) were shown in Figure S2, S3. Verification of the interaction between GSH and MnO2 NS/SC/AR The interesting mechanism observed above indicating that the mixture of MnO2 NS/SC/AR can be an ideal system for ratiometric detection of GSH, Scheme 1. Verification experiments were performed initially to study the interaction between GSH and MnO2 NS/SC/AR. As presented in Figure 3A, in the absence and presence of MnO2 NS, SC exhibited high and low FI465 signals (Figure 3A (a, b)), respectively, which were consistent with Figure 2B (S1/S2). Analogously, in the absence and presence of MnO2 NS, AR showed low and high FI585 signals (Figure 3B (a, b)), respectively. While, if certain amount of GSH was premixed with MnO2 NS, it will etch the nanosheet via reduction reaction (MnO2+2GSH+2H+=Mn2++GSSG+2H2O).20 Subsequently, an enhanced FI465 signal and an obviously decreased FI585 one were observed (Figure 3A (c), 3B (c)), respectively. To further identify mechanism of the interaction between GSH and MnO2 NS/SC/AR. The UV-vis spectra of MnO2 NS in the absence/presence of GSH were collected. With the addition of GSH, the characteristic peak at 380 nm of MnO2 NS decreased significantly, Figure 3C, suggesting the nanosheet’s sufficient decomposition. Besides, the interaction between pure Mn2+ and SC/AR was also conducted and we observed no obvious fluorescent changes of the two substrates, Figure 3D. All the above indicated that GSH is an efficient inhibitor of the oxidase-like property of MnO2 NS, which also supported the hypothesis that GSH-induced etching of MnO2 can be an effective probe for GSH’s detection.20



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Cascade logic circuit based on GSH and MnO2 NS/SC/AR As for the inherent intelligence/stringency of Boolean logic, molecular logic computing programmed analyses

37

28, 29

presents promising prospects for modular detection of targets and

intelligent diagnostics of diseases. In this work, molecular logic computation was integrated with ratiometric analysis of GSH for the first time. A cascade logic circuit (an INHIBIT logic gate cascade with a 1 to 2 decoder) was constructed based on above interaction between GSH and MnO2 NS/SC/AR, Figure 4A (the equivalent logic circuit). MnO2 NS and GSH were two inputs of the first INHIBIT gate, in which GSH played as the inhibitory element. For the output of the INHIBIT gate, the existence of complete MnO2 NS was defined as output “1” and other products (buffer, GSH, mixture of GSSG and Mn2+) were defined as “0”, respectively. Subsequently, the output of the INHIBIT gate was utilized as the input of downstream 1 to 2 decoder, in which the mixture of SC and AR was the platform. For the final output of the cascade logic circuit, the threshold value was set as “150” a.u. (short for arbitrary unit) to judge the positive/negative outputs, and the high and low FI465 (SC-ox) & FI585 (AR-ox) were implemented as “1”/“0” of the two outputs, respectively. As shown in Figure 4B, the fluorescent column bars of SC-ox (blue columns) and AR-ox (red columns) under different input variations featured the operating principle of the cascade logic circuit properly, indicating the successful construction of it. And the error bars fully proved the reproducibility of the results. Before the detection of GSH, an initial judgment for the absence/presence of sufficient GSH can be executed via the logical operations of above cascade logic circuit. Corresponding truth table was presented in Figure 4C, observed from the dual-mode output states of input variations “10” and “01” (denoted with purple square frame), the absence/presence of GSH can be readily distinguished. This cascade logic circuit endowed subsequent GSH sensing with the intelligence/stringency of Boolean logic.


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Ultrasensitive and selective detection of GSH. After the construction of the cascade logic circuit, ratiometric detection of GSH programmed by the logic circuit was executed, Scheme 1. In the absence of GSH, MnO2 NS will oxidize fluorescent SC and non-fluorescent AR into non-fluorescent SC-ox and high-fluorescent AR-ox, respectively. And we obtained a negligible FI465 signal of SC-ox and a largely enhanced FI585 signal of AR-ox. While, with the addition of GSH, FI465 values increased gradually, accompanied with gradual decrease of FI585 values. Relevant calibration curve of the ratiometric values of F585/F465 as a function of different concentrations of GSH was presented in Figure 5C. And a linear relationship between the F585/F465 ratios and logarithmic values of GSH’s concentrations was obtained in the range from 20 nM to 2000 nM (y=-3.064 log (x) +11.325, R2 = 0.997), inset of Figure 5C. The error bars fully proved the reproducibility of the results. According to the principle of S/N=3, the calculated detection limit was 6.7 nM, which was much lower than previously reported ratiometric fluorescent sensors for GSH, and also the lowest MnO2 NS-based fluorescent GSH sensor reported so far (Table 1). Thus, ultrasensitive detection of GSH based on the cascade logic circuit was accomplished. The selectivity of sensors plays the same significant role with their sensitivity. 53, 54 To explore the selectivity of above ratiometric sensor, different metal ions and other complex biomolecules were applied. As presented in Figure 5D, in the absence of any reactants (the control background sample), the column bars presented the highest F585/F465 value (blue column), and other non-target molecules also produced much higher ratios than target GSH, suggesting the weaker interactions between them and MnO2 NS. Although certain amounts of cysteine (Cys) and ascorbic acid (AA) induced low ratios to some extent, their concentrations (µM level) in



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biological environments are obviously lower than that of GSH (mM level).18-20, 54 Therefore, this system still presented well selectivity for GSH’s detection. Recovery tests in human serum samples Real sample application of biosensors could not only broaden the practical use of them but also lay the foundation for point-of-care test of diseases in the future.53, 54 For the real sample analysis, the diluted human serum samples were utilized to perform the recovery tests. As shown in Table 2, the obtained recoveries ranged from 97% to 101 %, which indicated the satisfactory applicability of this ratiometric fluorescent sensor in human serum samples. CONCLUSIONS In summary, we for the first time expanded the application of oxidase-like property of MnO2 NS to fluorescent substrates of peroxidase. Surprisingly, we found that MnO2 NS could not only largely quench the fluorescence of highly-fluorescent SC but also enhance that of nonfluorescent AR. Taking advantage of this phenomenon and the unique reaction between GSH and MnO2 NS, we constructed the first MnO2 NS-based ratiometric fluorescent sensor that programmed by a cascade logic circuit for ultrasensitive and selective detection of GSH. The calculated detection limit reached to as low as 6.7 nM, which was the lowest MnO2 NS-based fluorescent GSH sensor reported so far, and also much lower than previous ratiometric sensors for GSH. Furthermore, this sensor was simple, label-free and low-cost, it also presented excellent applicability in human serum samples. This study not only brought ratiometric fluorescence to MnO2 NS-based biosensing, pioneered the application of MnO2 NS’ oxidase-like property to fluorescent substrates, but also opened promising avenues for more powerful logical biosensors that operated upon other nanomaterials.


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EXPERIMENTAL SECTION Materials. KMnO4, MnCl2, KCl, NaCl and MgCl2 were obtained from Xilong Chemical Co. Ltd. (Guangdong, China); 2-(N-morpholino) ethane sulfonic acid (MES), GSH, Histidine (His), Phenylalanine (Phe), Lysine (Lys), Serine (Ser), Threonine (Thr), Aspartic acid (Asp), Glutamic acid (Glu), Cysteine (Cys) were purchased from Shanghai Sangon Biotechnology (Shanghai, China), Ascorbic acid (AA) was provided by Sigma-aldrich (USA). Scopoletin (SC) (98%) was provided by J&K (Beijing, China) and dissolved with dimethyl sulfoxide (DMSO) to 65 mM as stock solution. Amplex Red (AR) (≥98%) was obtained from Aladdin Industrial Corporation (Shanghai, China) and dissolved with DMSO to 16 mM as stock solution. 1×HEPES buffer (25 mM HEPES, 0.05% (w/v) Triton X-100, 1% (v/v) DMSO, pH 7.4) was used throughout the experiments and the distilled water was purified via a Millipore system. Synthesis of MnO2 NS. The synthesis of MnO2 NS was identical to previous study with minor modifications.20 Typically, 1 mM KMnO4 was added to 0.01 M MES buffer that dissolved with 10 mL distilled water. Then, the mixture was ultrasonicated for about 30 min. And MnO2 NS was obtained after centrifugation at 10000 rpm for 10 min. After washing with distilled water for five times, the final product was dispersed with 10 mL distilled water and stored at 4 °C as stock solution. The concentration of element manganese (Mn) in the stock solution was identified as 379.6 µM through ICP-MS measurement and used to quantify the level of MnO2 NS. Apparatus.



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Transmission electron microscopy (TEM) was taken by a JEM-2010 (HR) microscope operated at 200 kV. X-Ray photoelectron spectroscopy (XPS) was obtained from an ESCALAB-MKII Xray photoelectron spectroscope (VG Scientific, UK). Typical detection of GSH. For the typical detection of GSH, 200 µL above stock solution of MnO2 NS was mixed with different concentrations of GSH and reacted at room temperature for about 5 min. Then, 3 µL SC (250 µM) and 5 µL AR (250 µM) were added into the solution. Finally, suitable volume of HEPES buffer was added at last to make the total volume be 500 µL. After reacted at dark for about 15 min, the fluorescence of different samples were collected. Fluorescence Spectra Measurement. The fluorescence spectra of SC (excited at 380 nm) and AR (excited at 560 nm) in different samples were measured on a Cary 50 Eclipse Fluorescence Spectrophotometer (Agilent Technologies, USA). The excitation/emission slit widths for SC were 5/10 nm, respectively, and that for AR were 5/5 nm, respectively. Recovery tests of GSH in human serum samples. The human serum samples were diluted with HEPES buffer initially to make the concentrations of GSH in the linear range of detection. Then, a certain amount of GSH was added into one of the duplicate samples, after that the standard and test samples were used for recovery measurement. ASSOCIATED CONTENT


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The Supporting Information is available free of charge on the ACS Publications website at DOI: ……. AFM image of MnO2 NS, optimization of the amounts of MnO2 NS and oxidation time of fluorescent substrates. The following files are available free of charge. AUTHOR INFORMATION Corresponding Author * Email: [email protected]. Fax: +86-43185689711. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Nos. 21375123, 21427811 & 21675151). REFERENCES 1.

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Figures and Schemes

Scheme 1. (A) Illustration of the cascade logic circuit (INHIBIT-1 to 2 decoder); (B) schematic operations of the MnO2 NS-based ratiometric fluorescent sensor for GSH based on two fluorescent substrates. The fluorescent SC and non-fluorescent SC-ox (oxidized SC) are pictured by blue star with and without halo, respectively. And the non-fluorescent AR and fluorescent AR-ox (oxidized AR) are pictured by red star without and with halo, respectively.


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Figure 1. (A) TEM image of as-synthesized ultra-thin MnO2 NS; (B) UV-vis absorbance spectra of MnO2 NS and the photo of the suspension (inset); (C) XPS spectra of MnO2 NS; (D) Potential catalytic oxidation mechanism of SC and AR by MnO2 NS.



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Figure 2. (A) Fluorescence spectra of separately-used SC in the absence (a) and presence (b) of MnO2 NS and that of separately-used AR (c, d); (B) 3D fluorescent columns of simultaneouslyused SC (blue columns, S1, S2) and that of AR (red columns, A1, A2) without and with MnO2 NS.


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Figure 3. (A) Fluorescence spectra of simultaneously-used SC in the presence of no reactants (a), MnO2 NS (b) and the mixture of GSH/MnO2 NS (c); (B) Fluorescence spectra of simultaneously-used AR in the presence of no reactants (a), MnO2 NS (b) and the mixture of GSH/MnO2 NS (c); (C) UV-vis spectra of MnO2 NS in the absence (a) and presence (b) of GSH; (D) Fluorescence spectra of simultaneously-used SC in the absence (a) and presence (b) of Mn2+ and that of AR (c, d).



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Figure 4. (A) Equivalent logic symbol of the cascade logic circuit, an INHIBIT gate cascade with a 1 to 2 decoder, in which the FI465 of SC-ox and FI585 of AR-ox are two final outputs; (B) Fluorescent column bars of simultaneously-used SC-ox (blue columns) and AR-ox (red columns) under different input variations, “00” represents the absence of MnO2 NS and GSH; “01” represents the absence of MnO2 NS and presence of GSH, respectively, other ditto, the error bars are obtained via three independent experiments; (C) Truth table of the cascade logic circuit. (The concentration of GSH used in the cascade logic circuit is 10 µM).


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Figure 5. (A) Fluorescence spectra of SC in the presence of MnO2 NS and increasing concentrations of GSH; (B) Fluorescence spectra of AR in the presence of MnO2 NS and increasing concentrations of GSH; (C) Calibration curve between the ratiometric fluorescent values (FI585/FI465) as a function of the amounts of GSH, linear relationship between the ratios and logarithmic values of GSH’s concentrations that obtained in the range from 20 nM to 2000 nM (inset of Figure 5C), the error bars are obtained via three independent experiments.; (D) Ratiometric fluorescent columns with error bars in the presence of various reactants. The concentrations of various metal ions and His, Phe, Lys, Ser, Thr, Asp and Glu are all 100 µM, and that of GSH is 10 µM, the concentrations of Cys and AA are 1 µM. The error bars are obtained via three independent experiments.



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Table 1. Comparison between this GSH sensor and previously reported typical ratiometric fluorescent ones and other MnO2 NS-based ones.


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Table 2. Recovery test results of GSH in human serum samples.



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For TOC Only


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Introducing Ratiometric Fluorescence to MnO2 Nanosheet-Based

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