supporting information for · 8. examination of ecor i-triggered cleavage of f-pecor by...

Supporting information for

A general fluorescent sensor design strategy for “turn-on”

activity detection of exonucleases and restriction endonucleases

based on graphene oxide 1. Characterization of GO

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Fig. S1 Characterization of GO. (a) AFM image and (b) height profile of GO show

the dimensions of as-prepared GO as 0.1-0.3 μm in width and 0.98 nm in height. (c)

UV spectra and (d) FT-IR spectra of GO indicate characteristic peaks of oxygen

functional group. 2. Raman characterization of two GOs with different oxidation degrees

Electronic Supplementary Material (ESI) for AnalystThis journal is © The Royal Society of Chemistry 2013

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L-GO H-GO

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nsity

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Fig. S2 Raman spectra of two GOs with different oxidation degrees.


3. X-ray photoelectron spectroscopy (XPS) characterization of two GOs with different oxidation degrees

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Fig. S3 XPS wide spectra of L-GO(top) and H-GO (bottom).


4. Fluorescence quenching of fluorescent labeled ss-DNAs by the two GOs with different oxidation degrees

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1 (1) F-pExo(2) F-pExo + L-GO(3) F-pExo + H-GO

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1(1) F-pEcoR(2) F-pEcoR + L-GO(3) F-pEcoR + H-GO

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Fig. S4 Fluorescence emission spectra of fluorescent labeled ss-DNAs under different

conditions. (a) The fluorescent labeled F-pExo was used. [F-pExo] = 200 nM, [L-GO]

= 16 μg/100 μL, [H-GO] = 50 μg/100 μL; (b) The fluorescent labeled F-pEcoR was

used. [F-pEcoR] = 100 nM, [L-GO] = 8 μg/100 μL, [H-GO] = 20 μg/100 μL; (c) The

fluorescent labeled F-pHind was used. [F-pHind] = 100 nM; [L-GO] = 10 μg/100 μL;

[H-GO] = 25 μg/100 μL.


5. Thickness change of L-GO before and after Exo 1 treatment

Fig. S5 AFM images of the L-GO/F-pExo mixture before (A) and after (B) treatment

with Exo 1.


6. The morphology of GO in different conditions

The morphology of GO in different conditions was characterized by

high-resolution transmission electron microscopy (HRTEM). Before ultrasound

treatment, only large black spots could be observed, indicating that the synthesized

GOs aggregated together (Fig. S6A). However, after an ultrasound treatment,

wrinkled and silk–like sheets were observed on the top of the grid, which is the

typical HRTEM image of single-layered GO (Fig. S6B). The presence of fluorescent

labeled ss-DNA (F-pExo) and Exo 1 nearly had no effect on the morphology of the

L-GO. The large sheet still wrinkled but not folded (Fig. S6C,D).

Fig. S6 HRTEM images of GO in different conditions. (A) GO before ultrasound

treatment; (A) GO after ultrasound treatment; (C) The mixture of L-GO and F-pExo;

(D) The mixture of L-GO and F-pExo after treatment with Exo 1.


7. Optimization of digestion reaction time for Exo 1 detection

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Fig. S7 Digestion time-dependent fluorescence change of the sensing system. [F-pExo]

= 200 nM. Reaction temperature was 37°C.


8. Examination of EcoR I-triggered cleavage of F-pEcoR by polyacrylamide gel

electrophoresis (PAGE)

5 μL of 100 μM F-pEcoR solution was dissolved in 35 μL Tris-HCl buffer (pH = 7.5)

containing 50 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2 and 1 mM DTT. The

mixture was heated at 95 oC for 5 min, then cooled slowly to room temperature (25 oC)

and incubated at 25 oC for 30 min.. Different concentrations of EcoR I were added.

The final volume of the mixture was 50 μL. After incubation at 37 oC for 2 h, 5 μL

loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol, 10 mM

EDTA) was added. 30 μL of samples were loaded on 21% polyacrylamide gel.

Electrophoresis was carried out in 1 TBE buffer at 10 V/cm for 3h at room

temperature. The gel was photographed using a Gel Documentation system

(Huifuxingye, Beijing, China).

Fig. S8 PAGE analysis of EcoR I-dependent cleavage of F-pEcoR. Lane 1: F-pEcoR

only; Lane 2: F-pEcoR is treated with 40 U/100 L inactive EcoR I; Lane 3-6:

F-pEcoR is treated with 10, 20, 40 or 100 U/100 L EcoR I, respectively. [F-pEcoR]

= 10 M.


9. Feasibility of the proposed EcoR I sensor

Fig. S9 Fluorescence emission spectra under different conditions. The insert shows

the photographed images of the detection system in the absence or presence of EcoR I

when they are irradiated with light of 480 nm. [F-pEcoR] = 100 nM; [L-GO] = 8

μg/100 μL; [EcoR I] = 0.15 U/100 μL. The inactive EcoR I was prepared by heating

the enzyme solution at 95 oC for 20 min.


10. Optimization of L-GO concentration for EcoR I detection

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L-GO concentration (g/100 L)

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Fig. S10 Fluorescence signals of the sensing system containing different L-GO

concentrations in the absence or presence of 0.15 U/100 μL EcoR I. F/F0 is defined as

the signal-to-background ratio, F and F0 represent the fluorescence intensities in the

presence and absence of EcoR I, respectively. [F-pEcoR] = 100 nM. λex = 480 nm, λem

= 519 nm.


11. Optimization of digestion reaction time for EcoR I detection

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EcoR I activity: 0.5 U/100 L 2 U/100 L

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Fig. S11 Digestion time-dependent fluorescence change of the sensing system.

[F-pEcoR] = 100 nM. Reaction temperature was 37 °C.


12. Comparison of our EcoR I activity detection method with other reported methods

Table S1 Comparison of several EcoR I activity detection methods

Method Linear range (U/mL) Detection limit (U/mL) Reference

gold nanorods 1.0-100 0.65 17

Enzyme-responsive vanoparticle

25-150 Not given 11

Cationic conjugated polymer/DNA complexes

Not given 0.15 34

G-quadruplex DNAzyme-based fluorescent probe

0.1-5 0.1 35

SYBR Green I method 40 -300 8 36

This method 0.1-2 U/100 μL 0.06


13. Optimization of L-GO concentration for Hind III detection

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Fig. S12 Fluorescence signals of the sensing system containing different L-GO

concentrations in the absence or presence of 0.19 U/100 μL Hind III. F/F0 is defined

as the signal-to-background ratio, F and F0 represent the fluorescence intensities in the

presence and absence of Hind III, respectively. λex = 480 nm, λem = 519 nm.

According to the result, 10 μg/100 μL was selected as the optimal L-GO

concentration.


14. Hind III activity detection using the proposed Hind III sensor.

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Hind III activity (U/100 L) Fig. S13 Sensitivity of GO-based Hind III sensor. (A) Hind III activity-dependent

change in fluorescence spectra of the sensing system. The activities of Exo1 are

(arrow direction): 0, 0.01, 0.02, 0.04, 0.07, 0.12, 0.14, 0.17, 0.19, 0.20, 0.23, 0.26,

0.30 U/100 μL. (B) Hind III activity-dependent change in the fluorescence signal at

519 nm. The insert shows the absorption signal change in the Hind III activity range

of 0.01-0.30 U/100 μL. The solid line represents a linear fit to the data. All

experiments were performed in triplicate.


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