Mechanistic Study and Application for Sensitive Sensing of Ascorbic

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Single-Layer MnO2 Nanosheets Suppressed Fluorescence of 7-Hydroxycoumarin: Mechanistic Study and Application for Sensitive Sensing of Ascorbic Acid In Vivo Wanying Zhai, Chunxia Wang, Ping Yu, Yuexiang Wang, and Lanqun Mao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac503215z • Publication Date (Web): 13 Nov 2014 Downloaded from http://pubs.acs.org on November 17, 2014

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Analytical Chemistry

Single-Layer

MnO2

Nanosheets

Suppressed

Fluorescence

of

7-Hydroxycoumarin: Mechanistic Study and Application for Sensitive Sensing of Ascorbic Acid In Vivo

Wanying Zhai, Chunxia Wang, Ping Yu, Yuexiang Wang, Lanqun Mao* Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, the Chinese Academy of Sciences (CAS), Beijing 100190, China.

*

Corresponding Author. Fax: +86-10-62559373; Email: [email protected]

1

ACS Paragon Plus Environment


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Abstract. In this study, we systematically investigate the mechanism of single-layer MnO2 nanosheets suppressing fluorescence of 7-hydroxycoumarin and, based on this, demonstrate a new fluorescent method for in vivo sensing of ascorbic acid (AA) in rat brain. The mechanism for the fluorescence suppression is attributed to a combination of inner filter effect (IFE) and static quenching effect (SQE), which is different from those reported for the traditional two-dimensional nanosheets, and Förster resonant energy transfer (FRET) mechanism reported for MnO2 nanosheets. The combination of IFE and SQE leads to an exponential decay in fluorescence intensity of 7-hydroxycoumarin with increasing concentration of MnO2 nanosheets in solution. Such a property allows optimization of the concentration of MnO2 nanosheets in such a way that the addition of reductive analyte (e.g., AA) will to the greatest extent restore the MnO2 nanosheets-suppressed fluorescence of 7-hydroxycoumarin through the redox reaction between AA and MnO2 nanosheets. Based on this feature, we demonstrate a fluorescent method for in vivo sensing of AA in the cerebral systems with an improved sensitivity. Compared with the turn-on fluorescent method through first decreasing the fluorescence to the lowest level by adding concentrated MnO2 nanosheets, the method demonstrated here possesses a higher sensitivity, lower limit of detection, and wider linear range. Upon the use of ascorbate oxidase to achieve the selectivity for AA, the turn-on fluorescence method demonstrated here can be used for in vivo sensing of AA in a simple but reliable way.

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INTRODUCTION Two-dimensional (2D) nanosheets are of great interest because of their unique properties including exceptionally high specific surface area, rich structural diversity and unique electronic property.1-8 Recent research in such materials has become intense as a result of the advances in various techniques to produce different sorts of nanosheets such as graphene oxides, metal disulfides, metal oxides and carbon nitride.9-20 In this context, researchers have found that some nanosheets can serve as a novel platform for fluorescent sensing.21-26 Among these nanosheets materials, graphene oxides represent one kind of 2D carbon nanosheets obtained from acid exfoliation of graphite and have been widely employed in various applications.27-30 For example, graphene oxides have been successfully used for fluorescent sensing of different analyte ranging from nucleic acids, proteins, small molecules and metal ions.27-30 As one kind of redox active 2D nanomaterials, single-layer MnO2 nanosheets have three atomic layers, namely, one Mn layer sandwiched by two O layers.31 Each Mn coordinates to six O atoms to form an edge-sharing MnO6 octachedra. The existence of Mn-vacancies makes single-layer MnO2 nanosheets negatively charged and repulse each other.32 Due to this electrostatic repulsion, a stable colloidal suspension of single-layer MnO2 nanosheets could form, which is particularly useful for solution manipulation of single-layer MnO2 nanosheets to establish a platform for fluorescent sensing. Moreover, the d-d transition of Mn ions in the MnO6 octahedra of MnO2 nanosheets results in a broad absorption spectrum (~ 200-600 nm) with a large molar extinction coefficiency (εmax = 9.6 × 103 M-1cm-1).33 The spectrum overlaps with the fluorescence excitation and/or emission spectra of most kinds of organic dyes. This feature allows the occurrence of FRET34 and possible IFE with single-layer MnO2 nanosheets, of which FRET mechanism has recently been utilized for fluorescent sensing.35 On the other hand, different from other kinds of 2D 3



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nanosheets, MnO2 nanosheets themselves have strong oxidation ability and can oxidize many organic compounds. Such a property can also be used to constitute a sensing strategy for practical applications.36 In this study, we systematically investigate the mechanism for single-layer MnO2 nanosheets suppressing fluorescence of 7-hydroxycoumarin. Interestingly, we find that the fluorescence suppression of 7-hydroxycoumarin is based on the combination of IFE and static quenching effect (SQE), which is relatively different from those reported for the traditional 2D nanosheets such as graphene oxides27-30 and from the FRET mechanism recently reported for single-layer MnO2 nanosheets.36-38 The mechanism (i.e., IFE-SQE) is essentially originated from the unique properties of single-layer MnO2 nanosheets described above; MnO2 nanosheets exhibit a broad absorption spectrum with a large molar extinction coefficient and rich surface chemistry, which enable the co-occurrence of IFE and SQE on the fluorescence of 7-hydroxycoumarin used in this study with the presence of MnO2 nanosheets. Based on this mechanism, we develop a new fluorescent method for in vivo sensing of AA with an improved sensitivity through the exponential decay in the fluorescence of 7-hydroxycoumarin with the presence of MnO2 nanosheets (Scheme 1). The exponential decay feature allows the optimization of the concentration of MnO2 nanosheets in such a way that the fluorescence decay is to the greatest extent sensitive to the change of the concentration of MnO2 nanosheets, ensuring a high sensitivity and low limit of detection for AA sensing since AA can chemically reduce and thus consume MnO2 nanosheets. This property, combined with the high selectivity of ascorbate oxidase to selectively catalyze the oxidation of AA, enables a turn-on fluorescent method for sensitive and selective sensing of AA in the microdialysate from rat brain. Although MnO2 nanosheets have recently been used for biomolecular sensing with FRET mechanism,36-38 the mechanism demonstrated here for the fluorescence suppression of 7-hydroxycoumarin and its application to constitute a 4


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fluorescent method for in vivo sensing of AA have not been reported so far.

H

H OH HO H

OH OH

HO

Mn O

HO

O Mn

HO

O

O O

H

O

Mn O

Mn HO

Mn O

O

HO

HO

O

O Mn O

OH

OH HO O Mn O

O

O

O

O

O

OH

HO

OH

O

OH

O Mn

O

H

OH

O Mn O

OH

H

2+

OH

OH H

Mn

Mn O

O

O

H O

O Mn

Mn

OH

H

OH

O

AA

OH Mn

O O

O Mn

OH O

Mn O

O Mn

O

Mn

HO

OH

Mn O

H OH

O Mn

O O

Mn

H OH

O Mn

O O

O

Mn HO

H

Mn

Mn O

H

O Mn

O O

H OH

HO

O O

HO

O

OH OH

Mn

H HO

OH

O

OH H

IFE + SQE

Mn

O Mn

OH H

H O

O

O O

O OH

OH Mn

O Mn

O Mn

OH OH

H

O

O

OH O

Mn O

Mn

O

Mn O

H OH

O Mn

O O

O

H OH

O Mn

O

Mn

O

H OH

O Mn

O

OH H

H

H OH HO

OH OH

HO

Mn

H O HO Mn HO

Mn O

O H

O O Mn

O Mn

HO

Mn

O

O

O HO

OH OH

H

HO

O

O O

O H

O

O

OH O

Mn O

OH H

OH Mn

O

Mn

OH

H O

O Mn

Mn

OH

OH Mn

O O

O Mn

OH O

Mn O

O Mn

O

Mn

H OH

O Mn

O O

O

H OH

O Mn

O O

O

H OH

O Mn

O O

H OH

OH H

MnO2 Nanosheets

7-Hydroxycoumarin

Scheme 1. In Vivo Fluorescent Sensing of AA through MnO2 Nanosheets Suppressed Fluorescence of 7-Hydroxycoumarin with the Combination of IFE and SQE.

EXPERIMENTAL SECTION Chemicals and Solutions. 7-Hydroxycoumarin (99%) was purchased from Aldrich and used as received. Ascorbic acid (AA) and ascorbate oxidase (AAOx) (Cucurbita species, EC 1.10.3.3) were purchased from Sigma and were used as received. Sodium pyrophosphate (PPi) was purchased from Beijing Chemical Reagent Co. (Beijing, China). Artificial cerebrospinal fluid (aCSF) used as a perfusion solution for in vivo microdialysis was prepared by mixing NaCl (126 mM), KCl (2.4 mM), KH2PO4 (0.5 mM), MgCl2 (0.85 mM), NaHCO3 (27.5 mM), Na2SO4 (0.5 mM) and CaCl2 (1.1 mM) into Milli-Q water. All aqueous solutions were prepared with Milli-Q water (18.2 MΩ cm). No buffer solution was used in the fluorescent quenching experiment. Preparation of Single-Layer MnO2 Nanosheets. Single-layer MnO2 nanosheets were prepared as reported previously.33 In a typical synthesis, 20 mL of a mixture containing 12 mL tetramethylammonium (TMA, 1.0 M in H2O) and 2 mL H2O2 (30 wt% in H2O) was added into 10 mL of the aqueous solution of 0.593 g MnCl2•H2O (99.99%) within 15 s. The as-prepared dark brown suspension was stirred vigorously overnight at room temperature in the open air. The thickness of as-formed single-layer MnO2 nanosheets was characterized to 5



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be ~ 0.9 nm by the tapping-mode atomic force microscopy (Figure S1), confirming that a single-layer MnO2 nanosheet was obtained. To calculate the concentration of single-layer MnO2 nanosheets in water, the absorption spectra of the as-prepared single-layer MnO2 nanosheets were measured and the concentration was calculated according to Lambert-Beer’s Law with the molar extinction coefficient of 9.6 × 103 M-1cm-1 at 380 nm.33 Instrumentation.

Absorption spectra of single-layer MnO2 nanosheets and

7-hydroxycoumarin were recorded on a TU 1900 spectrometer (Beijing, China). Photoluminescence spectroscopy was measured on a Hitachi F-4600 spectrometer (Hitachi Co. Ltd., Japan) with Xe lamp as the excitation source. Time-resolved fluorescence experiments were carried out on a FLS920 combined with fluorescence lifetime and steady-state spectrometer (Edinburgh Co. Ltd., England). The thickness of MnO2 nanosheets was measured with atomic force microscopy (AFM, Dimensional 3100, Veeco Co). In Vivo Microdialysis and AA Sensing. In vivo microdialysis was performed with the procedures reported in our previous works.39-40 In brief, adult male Sprague-Dawley rats (190-300 g, obtained from Health Science Center, Peking University) were housed on a 12:12 h light-dark schedule with food and water ad libitum. The animals were anaesthetized with pentobarbital (345 mg kg-1, i.p.) and then positioned onto a stereotaxic frame. The microdialysis guide cannulas were implanted into the striatum (AP = 0 mm, L = 3 mm from bregma; V = 4.5 mm from dura) using a standard stereotaxic procedure.41 The guide cannula was kept in place with three skull screws and dental cement. Stainless steel dummy blockers were inserted into the guide cannula and fixed until the insertion of the microdialysis probe. Throughout the surgery, the body temperature of the animals was maintained at 37 °C with a heating pad. Immediately after surgery, the animals were put into a warm incubator individually until they recovered from the anesthesia. After the animal recovered for at least 24 h, a microdialysis probe (CMA, dialysis length, 4 mm; diameter, 0.24 mm) was implanted 6


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into the rat striatum and then perfused with aCSF via FEP tubing pumped at a flow rate of 3 µL min-1 for at least 90 min for equilibration. After that, the microdialysate was collected in an ice bath for immediate analysis. For fluorescent sensing of AA in the brain microdialysate in vivo sampled from rat striatum, a reaction mixture containing 7-hydroxycoumarin (50 µM), MnO2 nanosheets (100 µM) and pyrophosphate ion (PPi, 100 µM) was first prepared and the photoluminescence spectrum of the reaction mixture was recorded immediately. The collected microdialysate (20 µL) was divided into two parts with equal volume. One part (10 µL) was directly added into the reaction mixture (40 µL) and the resulting mixture was first incubated in a 25°C water bath for 3 min and then its photoluminescence spectroscopy was measured. To the other part of the microdialysate (10 µL), 1 µL of AAOx (250 U/mL) was added and the mixture was then allowed to stand by for 20 min in a 25 °C water bath. After that, the mixture was added into the reaction mixture (40 µL) mentioned above and the resulting mixture was first incubated in a 25 °C water bath for 3 min and then its photoluminescence spectroscopy was measured. The concentration of AA in the brain microdialysate was determined by subtracting the fluorescence intensity at emission wavelength of 466 nm of the reaction mixture containing AAOx-treated microdialysate with that of the reaction mixture containing the untreated microdialysate.

RESULTS AND DISCUSSION Mechanism of MnO2 Nanosheets Suppressed Fluorescence of 7-Hydroxycoumarin. As displayed in Figure 1A, the fluorescence of 7-hydroxycoumarin decreases gradually with increasing the concentration of MnO2 nanosheets in solution. Since MnO2 nanosheets have a broad absorption spectrum in the range of 200-600 nm (Figure 1B, red curve), which overlaps with the fluorescence excitation and emission of 7-hydroxycoumarin (Figure 1B, 7



blue and black curves), the MnO2 nanosheets-induced fluorescence suppression of 7-hydroxycoumarin was first considered to stem from IFE. On the basis of the cuvette geometry used in the fluorescent measurements (Figure 2A) and of the absorption characteristics of the mixture of MnO2 nanosheets and 7-hydroxycoumarin, the IFE was estimated with eq. 1.42 2.3 2.3 = 10 1 − 10− 1 − 10−

(1)

Where, Fobsd is the measured maximum fluorescence intensity and Fcor is the corrected maximum fluorescence intensity by removing IFE from Fobsd; Aex and Aem represent the absorbance at the excitation wavelength (λex = 327 nm) and maximum emission wavelength (λem = 466 nm), respectively; s is the thickness of excitation beam (0.10 cm), g is the distance between the edge of the excitation beam and the edge of the cuvette (0.40 cm in this 1.0

MnO2

3000

2000

1000

Excitation

B)

1.0

Absorption Emission

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

Normalized F. I. / a. u.

A) Normalized Abs. / a. u.

4000

F. I./ a. u.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0 350

400

450

500

550

600

0.0 200

650

Wavelength / nm

300

400

500

600

0.0 700

Wavelength / nm

Figure 1. (A) Fluorescence spectra of 7-hydroxycoumarin (50 µM) with the addition of different concentrations of MnO2 nanosheets. λex = 327 nm. The concentration of MnO2 nanosheets was increased from (from upper to bottom) 0.0, 12.5, 25, 37.5, 50.0, 62.5, 75, 87.5, 100.0, 112.5, 125.0, 137.5, 150.0, 162.5, 175.0, 187.5, 200.0, 212.5, 225.0, 237.5, 250.0, 262.5, 275.0, to 287.5 µM. (B) Normalized fluorescence excitation (blue curve) and emission (black curve) spectra of 7-hydroxycoumarin and UV-Vis absorption spectrum of MnO2 nanosheets (red curve).

8


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A)

B) d

Cuvette

80 Observed Corrected

60

E%

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s g

40

20

F 0

10

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CMnO2 / µM

Figure 2. (A) The parameters used in eq. 1. (B) Suppressed efficiency (E %) of observed (black curve, ●) and corrected (red curve, ○) measurements for 7-hydroxycoumarin (50 µM) after each addition of different concentrations of MnO2 nanosheets. E % = 1 - F/F0. F0 and F are the steady-state fluorescence intensities of 7-hydroxycoumarin in the absence and presence of MnO2 nanosheets, respectively.

case), and d is the width of the cuvette (1.00 cm). The maximum value of the correction factor could not exceed 3; otherwise the correction was not convincing. Table 1 summarizes the concentrations of MnO2 nanosheets, absorbance and fluorescence intensity of 7-hydroxycoumarin after each addition of MnO2 nanosheets. The correction factor (CF) of IFE at each concentration of MnO2 nanosheets was calculated. After removing the IFE from the totally observed suppressed fluorescence, the suppressed efficiency for the totally observed and the corrected (i.e., after removing IFE) fluorescence of 7-hydroxycoumarin was figured out, as demonstrated in Figure 2B. We found that approximately half of the suppressed effect came from the IFE of MnO2 nanosheets. After removing the IFE, the remaining suppressed effect may come from the quenching effect of MnO2 nanosheets toward 7-hydroxycoumarin.

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Table 1 IFE of MnO2 Nanosheets on the Fluorescence of 7-Hydroxycoumarin MnO2 (µM) 0 10

Aex [a] 0.058 0.148

Aem [b] 0.001 0.049

CF [c] 1.06 1.24

Fobsd [d] 1101 886.6

Fcor [e] 1167.1 1099.4

Fcor,0/Fcor [f] 1 1.06

20 30 40 50 60

0.242 0.333 0.422 0.51 0.617

0.099 0.148 0.207 0.257 0.313

1.44 1.66 1.93 2.21 2.58

714.2 569.6 456.7 376.6 311.1

1028.4 945.5 881.4 832.3 804.0

1.13 1.23 1.32 1.40 1.45

[a]

Aex is the absorbance of 7-hydroxycoumarin with the addition of MnO2 nanosheets at

327 nm.

[b]

Aem is the absorbance of 7-hydroxycoumarin with the addition of MnO2

nanosheets at 466 nm. [c] Corrected factor (CF) was calculated as Fcor/Fobsd.

[d]

Fobsd is the

measured fluorescence intensity of 7-hydroxycoumarin with the addition of MnO2 nanosheets at 466 nm.

[e]

Fcor is the corrected fluorescence intensity with eq. 1 by

removing IFE from the measured fluorescence intensity (i.e., Fobsd).

[f]

Fcor,0 and Fcor are

the corrected fluorescence intensities of 7-hydroxycoumarin in the absence and presence of MnO2 nanosheets, respectively.

In general, fluorophores can be quenched through dynamic quenching effect (DQE) or static quenching effect (SQE) or both simultaneously.34 In DQE, the excited-state fluorophore is non-radiatively deactivated upon collision with the quencher. In SQE, the fluorophore forms a nonfluorescent complex with the quencher.34 When this complex absorbs light, it immediately returns to the ground-state without photon emission. Both DQE and SQE through ground-state complex formation model could be theoretically described by Stern-Volmer equation (i.e., eq. 2). 34 0 ⁄ = 1 +

(2)

Where, [Q] is the concentration of quencher, i.e., MnO2 nanosheets in this study; F0 and F are the steady-state fluorescence intensities in the absence and presence of the quencher,

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respectively. In our study, the corrected fluorescence intensity ratio (i.e., Fcor,0 /Fcor, Table 1) of 7-hydroxycoumarin in the absence and presence of MnO2 nanosheets was linear with the concentration of MnO2 nanosheets (i.e., CMnO2), as depicted in Figure 3 (Fcor,0 /Fcor = 0.008 CMnO2 / µM + 0.99, R2 = 0.994), suggesting that the Stern-Volmer equation (eq. 2) should be applied in this case and the Stern-Volmer quenching constant Ksv = 0.008 µM -1(as taken from the slope of the line in Figure 3).

1.5 1.4

Fcor,0 / Fcor

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1.3 1.2 1.1 1.0 0

10

20

30

40

50

60

CMnO2 / µM

Figure 3. Stern-Volmer plot for the fluorescence of 7-hydroxycoumarin quenched by different concentrations of MnO2 nanosheets after the IFE correction. Fcor,o and Fcor were the corrected fluorescence intensity of 7-hydroxycoumarin in the absence and presence of MnO2 nanosheets, respectively. The data were taken from Table 1.

Since both DQE and SQE fit into Stern-Volmer equation and thus could not be simply differentiated by this equation, the time-resolved fluorescence was then measured to clarify which type of quenching effect was involved in the fluorescence quenching observed in this study. The excited state lifetime for static quenching effect remains constant, whereas in the case for dynamic quenching effect the ratio τ0/τ of the life time is proportional to the ratio of F0/F. Figure 4 shows the typical fluorescence decay curves for 7-hydroxycoumarin in the absence and presence of MnO2 nanosheets. Before the addition of MnO2 nanosheets, the 11



2000 1500

Counts

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1000 500 0 0

10

20 Time / ns

30

40

Figure 4. Decay curves of 7-hydroxycoumarin (50 µM) in the absence (black curve) and presence (red curve) of MnO2 nanosheets (100 µM). λex = 340 nm, and λem = 455 nm. decay time constant τ0 was 5.35 ns. The presence of MnO2 nanosheets only leads to a very small change of τ (5.34 ns), suggesting that the fluorescence quenching observed above could be mainly due to SQE, rather than DQE. In SQE, a nonfluorescent complex is formed. However, in DQE, the quencher must diffuse to the fluorophore during the lifetime of its excited state and nonradiatively deactivate that state. Owing to the different affinity between fluorophore and quencher in SQE and DQE, the Stern-Volmer quenching constant (Ksv) is further analyzed to determine which type of quenching was being observed. In SQE, the Stern-Volmer quenching constant (Ksv) is interpreted as ground-state association constant for 7-hydroxycoumarin binding to MnO2 nanosheets. However, for DQE, the Stern-Volmer quenching constant (Ksv) is in the following equation,34

= × !0

(3)

Where, kq is the molecular quenching rate constant, and τ0 is the fluorescence lifetime in the absence of MnO2 nanosheets. For 7-hydroxycoumarin, Ksv = 0.008 µM -1 and τ0 = 5.35 ns. kq was then calculated to be 1.49 × 1012 M-1s-1. This value was unreasonably high because the

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largest possible value for a molecule in a diffusion-controlled process in aqueous solution is around 1 × 1010 M-1s-1.34 Thus, the DQE might not be possibly responsible for the observed quenching process. The analysis above again leads to a conclusion that the observed quenching effect, after the correction of IFE, could be mainly resulted from SQE. Since energy transfer has been normally considered as the common mechanism for the decreased fluorescence intensity43 when 2D nanomaterials, such as graphene oxides27 and MnO2 nanosheets,36-38 were used, we then studied the role of Förster resonant energy transfer (FRET) in the entire suppression process in our system. We found that FRET is only a tiny part in the entire suppression process, since the maximum efficiency it could reach was just about 6% (Supporting Information). Thus, the mechanism of both IFE and SQE presumably dominates the whole suppression process of 7-hydroxycoumarin caused by single-layer MnO2 nanosheets. Towards In Vivo Fluorescent Sensing of AA in Rat Brain. As described above, the suppressed fluorescence of 7-hydroxycoumarin caused by MnO2 nanosheets was based on the combination of IFE and SQE. Such a mechanism leads to an exponential decay in fluorescence intensity of 7-hydroxycoumarin with increasing the concentration of MnO2 nanosheets in solution, as shown Figure 5A. The exponential decay in Figure 5A was fitted into a linear plot (eq. 4, R2=0.998) (Figure 5B). "#$ %2 = 0.01622 ln(0 ⁄ )

(4)

Where, CMnO2 is the concentration of MnO2 nanosheets; F0 and F represent the observed fluorescence intensity of 7-hydroxycoumarin in the absence and presence of MnO2 nanosheets, respectively. The plot in Figure 5B was further derived into eq. 5 and the relationship between dF/dCMnO2 and CMnO2 was shown in Figure 5C.

13



= +$( ⁄0 )⁄"#$ %2 "#$ %2

(5)

As shown in Figure 5C, the higher concentration of MnO2 nanosheets used, the closer to zero of the derivative value of dF/dCMnO2. This suggests that, when a high concentration of MnO2 nanosheets was used to suppress the fluorescence of 7-hydroxycoumarin, the change of the fluorescence of 7-hydroxycoumarin is less sensitive to the change of the concentration of MnO2 nanosheets, as compared with the case when a low concentration of MnO2 nanosheets was used. This feature allows us to rationally optimize the concentration of 120

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∆F

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CMnO2 / µM

CMnO2 / µM

Figure 5 (A) Fluorescence change of 7-hydroxycoumarin (at λem= 466 nm) with various concentrations of MnO2 nanosheets (F and F0 represent the fluorescence intensity of 7-hydroxycoumarin in the presence and absence of MnO2 nanosheets, respectively). (B) Fitting curve of the plot in Figure 5A. (C) Derivative of the fluorescence of 7-hydroxycoumarin against the concentration of MnO2 nanosheets. (D) Recovery of 7-hydroxycoumarin fluorescence (50 µM, λex = 327 nm, λem = 466 nm) as a function of the concentration of MnO2 nanosheets when 2 µM AA was used as the analyte. 14


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MnO2 nanosheets to achieve a high sensitivity for the turn-on fluorescent sensing of the analyte based on the redox reaction between the reductive analyte (e.g., AA in this study) and MnO2 nanosheets. According to Figure 5C, to achieve a high sensitivity, the concentration of MnO2 nanosheets should be as low as possible. However, the background fluorescence signal would be strong if too low concentration of MnO2 nanosheets is used. Thus, an appropriate concentration of MnO2 nanosheets remains essential to achieve both purposes for fluorescent sensing. Here, we used 2 µM AA as an example to optimize the concentration of MnO2 nanosheets. As shown in Figure 5D, We found that an appropriate background signal and considerable high sensitivity were achieved when the concentration of MnO2 nanosheets was 100 µM with the concentration of 7-hydroxycoumarin being 50 µM. To optimize the concentration of MnO2 nanosheets for a fluorescent sensor when MnO2 nanosheets were used as both the fluorescence suppresser and chemical oxidant for AA sensing, the linearity, detection limit and sensitivity were studied with the uses of 100 µM and 300 µM MnO2 nanosheets. Note that, to avoid the possible flocculation of MnO2 nanosheets resulted from Mn2+ or Mn3+ produced in the redox reaction employed for AA sensing, pyrophosphate ion (PPi) was added to the reaction system since it readily forms complexes with many metal cations, such as Ca2+, Mg2+, Mn3+, Mn2+ and Cu2+, and could thus avoid the flocculation of MnO2 colloid (Figure S3). As shown in Figure 6A, with the addition of AA, the fluorescence of 7-hydroxycoumarin pre-suppressed by 100 µM MnO2 nanosheets was restored and the restored fluorescence intensity (∆F = F-F0, where F and F0 represent the fluorescence intensity in the presence and in the absence of AA, respectively) was linear with the concentration of AA within a concentration range from 0.5 to 40 µM (∆F = 25.94CAA/µM + 43.93, R2 = 0.979, as shown in Figure 6B). The limit of detection (LOD) was 0.09 µM. 15



2000

F. I. / a. u.

1500

1000

500

1200

B)

1000 800

∆F

0 µM 0.5 µM 4 µM 8 µM 10 µM 15 µM 20 µM 25 µM 30 µM 35 µM 40 µM

A)

600 400 200

0 350

400

450

500

550

600

0

650

0

5

10

Wavelength / nm

120

0 µM 10 µM 20 µM 30 µM 40 µM 50 µM

C)

90

15

20

25

30 35 40 45

CAA / µM 120

60

D)

100 80

∆F

F. I. / a. u.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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60 40

30

20

0 350

400

450

500

550

600

0

650

Wavelength / nm

10

20

30

40

50

CAA / µM

Figure 6. Fluorescence spectra of 7-hydroxycoumarin (50 µM) in the presence of MnO2 nanosheets with different concentrations of 100 µM (A) and 300 µM (C), and PPi (100 µM) restored by different concentrations of AA. Figure 6B and 6D are the linear relationship between the enhanced fluorescence intensity and the concentration of AA for Figure 6A and 6C, respectively. The concentrations of AA were indicated in the figure. λex = 327 nm. Meanwhile, the response of AA in 300 µM MnO2 nanosheets mixture was displayed in Figure 6C and 6D. In this case, a large amount of MnO2 nanosheets (i.e., 300 µM) could suppress the fluorescence of 7-hydroxycoumarin to a much lower level than by 100 µM MnO2 nanosheets (Figure S4). Similar to the results obtained with 100 µM MnO2 nanosheets (Figure 6A), the addition of AA into the mixture containing 300 µM MnO2 nanosheets also restores the fluorescence of 7-hydroxycoumarin, as shown in Figure 6C, and the restored fluorescence intensity (i.e., ∆F) was linear with the concentration of AA within a concentration range from 10 to 50 µM (∆F = 2.76CAA/µM -23.30, R2 = 0.99, as shown in 16


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Figure 6D) with a LOD of 1.6 µM. The comparison of the analytical properties (Table 2) of two approaches reveals that the approach using 100 µM nanosheets was advantageous in terms of its wider linear range, higher sensitivity, and lower LOD.

Table 2. The Comparison of the Analytical Properties of Two Approaches Linear range (µM)

LOD (µM)

Sensitivity (µM-1)

100 µM MnO2

0.5-40

0.09

25.9

300 µM MnO2

10-50

1.6

2.7

As shown in Table 2, with optimized amount of MnO2 nanosheets, the method developed here exhibits excellent analytical properties, which validate the method for in vivo sensing of AA. To achieve the selectivity for in vivo sensing of AA, ascorbate oxidase (AAOx) was used to sort out the response of AA from the total response generated from the reductive species in rat brain such as glutathione and cysteine. To sense AA in vivo, two aliquots (10 µL each) of same brain microdialysates with and without AAOx treatment were added into 40 µL of the aqueous dispersions of 7-hydroxycoumarin containing MnO2 nanosheets. By calculating the difference between the fluorescent intensities obtained without and with AAOx (blue and red curves, Figure 7), the basal level of AA in rat brain microdialysates could be selectively detected, which was estimated to be 8.7 ± 2.5 µM (n = 3). This value was almost consistent with those reported in our earlier studies by the electrochemical methods 44-45. More importantly, compared with the microelectrode method for in vivo voltammetric measurement of ascorbic acid in rat brain,46 our method also possesses a much lower limit of detection.

17



800 600

F. I. / a. u.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 25

400 200 0 400

450

500

550

600

650

Wavelength / nm

Figure 7. Typical fluorescence spectra of 7-hydroxycoumarin (50 µM) containing 100 µM PPi after successive addition of 100 µM MnO2 nanosheets (black curve), brain microdialysate (blue curve), and a mixture of brain microdialysate and 250 U/mL AAOx (red curve). λex = 327 nm.

CONCLUSIONS We have investigated the mechanism for MnO2 nanosheets suppressing fluorescence of 7-hydroxycoumarin and found that the fluorescence suppression is based on the combination

of inner filter effect (IFE) and static quenching effect (SQE), which is different from those reported for the traditional two-dimensional nanosheets, and Förster resonant energy transfer (FRET) mechanism reported for MnO2 nanosheets. The combination of inner filter effect (IFE) and static quenching effect (SQE) is found to result in an exponential decay in the fluorescence intensity of 7-hydroxycoumarin with increasing concentration of MnO2 nanosheets in solution. By taking advantages of this property and oxidative property of MnO2 nanosheets toward AA, we have developed a new fluorescent method for in vivo sensing of AA in the cerebral systems. Compared with the turn-on fluorescent methods based on general strategy by first decreasing the fluorescence to the lowest values and then turning the fluorescence on, the method demonstrated here shows wider linear range, higher sensitivity, and lower limit of detection. Upon the use of ascorbate oxidase to achieve the 18


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selectivity for AA, the fluorescence method demonstrated here can be used for in vivo sensing of AA. This study not only offers a new avenue to in vivo sensing of AA but also provides a new insight into MnO2 nanosheets suppressing fluorescence of coumarins dyes.

19



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ACKNOWLEDGMENTS This research is financially supported by the NSF of China (Grant no. 20935005, 21127901, 21210007, and 91213305 for L.M. and 91132708 for P.Y.), the National Basic Research Program of China (973 programs, 2010CB33502 and 2013CB933704; 863 programs, 2010AA06Z302), and The Chinese Academy of Sciences (KJCX2-YW-W25).

ASSOCIATED CONTENT Supporting Information Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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For TOC only

H

H OH HO H

OH OH

HO

Mn O

HO

O Mn

HO

O

O O

H

O

Mn O

Mn HO

O

O

HO

HO

O

Mn O

OH

OH H

H

O

Mn

O

O

OH

HO

OH

O OH

H

OH

OH

O

Mn

Mn

O

H

OH

O O O

OH

H OH

OH H

Mn

Mn

O O

Mn

H O

O Mn

O

AA

OH Mn

O O

O Mn

OH O

Mn O

O Mn

O

Mn

HO

OH

O

H OH

O Mn

O O

Mn

O

H OH

O Mn

O O

Mn

O

HO

H

Mn

O

H

O Mn

O O

Mn

H OH

HO

O O

O

OH

OH OH

Mn O

HO

O

O

OH HO H HO Mn

OH

O

Mn O

IFE + SQE

Mn

O

O

H O

O

O

O

OH

OH Mn

O Mn

O Mn

OH OH

H

O

O

OH O

Mn O

Mn

O

Mn O

H OH

O Mn

O

Mn O

O

H OH

O Mn

O

Mn

O

H OH

O Mn

O

OH H

H

H OH HO

OH OH

HO

Mn

H O HO HO

O

O

Mn HO

O

O

O HO

OH OH

H

O O

Mn O

OH H

O

O

OH O

Mn O

OH H

OH Mn

O

Mn

OH

H O

O Mn

O

HO

OH Mn

O O

O

Mn

OH O

Mn O

O Mn

O

Mn

H OH

O Mn

O O

Mn O

H OH

O Mn

O O

Mn

O

O H

O O

Mn

H OH

O Mn

O O

Mn

H OH

OH H

MnO2 Nanosheets

7-Hydroxycoumarin

25


2+

Mechanistic Study and Application for Sensitive Sensing of Ascorbic

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