Upload
others
View
2
Download
0
Embed Size (px)
Citation preview
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
200 300 400 500 600 700 800
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4(c)
Abs
orba
nce
wavelength(nm)4000 3500 3000 2500 2000 1500 1000 500
20
40
60
80
100
Tran
smis
sion
Wavelength(nm)
3409
1730
1625
1403
1224
1074
(d)
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
1100 1200 1300 1400 1500 1600 1700 1800
L-GO H-GO
Inte
nsity
(a.u
.)
Raman shift(cm-1)
Fig. S2 Raman spectra of two GOs with different oxidation degrees.
Electronic Supplementary Material (ESI) for AnalystThis journal is © The Royal Society of Chemistry 2013
3. X-ray photoelectron spectroscopy (XPS) characterization of two GOs with different oxidation degrees
400 600 800 1000 1200 14000
20000
40000
60000
80000
100000
Binding energy(eV)
Inte
nsity
(a.u
.) C1s
O1s
L-GO
400 600 800 1000 1200 14000
20000
40000
60000
80000
100000
Binding energy(eV)
Inte
nsity
(a.u
.)
C1s
O1s
H-GO
Fig. S3 XPS wide spectra of L-GO(top) and H-GO (bottom).
Electronic Supplementary Material (ESI) for AnalystThis journal is © The Royal Society of Chemistry 2013
4. Fluorescence quenching of fluorescent labeled ss-DNAs by the two GOs with different oxidation degrees
500 520 540 560 580 600 620 640
0
50
100
150
200
250
300(a)
Wavelength (nm)
Fluo
resc
ence
3
2
1 (1) F-pExo(2) F-pExo + L-GO(3) F-pExo + H-GO
500 520 540 560 580 6000
10
20
30
40
50
60
70(b)
Wavelength (nm)
Fluo
resc
ence 3
2
1(1) F-pEcoR(2) F-pEcoR + L-GO(3) F-pEcoR + H-GO
500 520 540 560 580 6000
10
20
30
40
50
60
70
80
90(c)
Wavelength (nm)
Fluo
resc
ence
3
2
1 (1) F-pHind(2) F-pHind + L-GO(3) F-pHind + H-GO
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.
Electronic Supplementary Material (ESI) for AnalystThis journal is © The Royal Society of Chemistry 2013
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.
Electronic Supplementary Material (ESI) for AnalystThis journal is © The Royal Society of Chemistry 2013
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.
Electronic Supplementary Material (ESI) for AnalystThis journal is © The Royal Society of Chemistry 2013
7. Optimization of digestion reaction time for Exo 1 detection
0 20 40 60 80 100 120
0
50
100
150
200
250
300
350
Exo 1 activity: 0.5 U/100 L 1 U/100 L 5 U/100 L
Fluo
resc
ence
Reaction time (min)
Fig. S7 Digestion time-dependent fluorescence change of the sensing system. [F-pExo]
= 200 nM. Reaction temperature was 37°C.
Electronic Supplementary Material (ESI) for AnalystThis journal is © The Royal Society of Chemistry 2013
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.
Electronic Supplementary Material (ESI) for AnalystThis journal is © The Royal Society of Chemistry 2013
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.
Electronic Supplementary Material (ESI) for AnalystThis journal is © The Royal Society of Chemistry 2013
10. Optimization of L-GO concentration for EcoR I detection
0 2 4 6 8 100
102030405060708090
100
Fluo
resc
ence
L-GO concentration (g/100 L)
0 2 4 6 8 1 002468
1 01 21 4
F/F 0
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.
Electronic Supplementary Material (ESI) for AnalystThis journal is © The Royal Society of Chemistry 2013
11. Optimization of digestion reaction time for EcoR I detection
0 20 40 60 80 100 1200
20
40
60
80
100
Reaction time (min)
EcoR I activity: 0.5 U/100 L 2 U/100 L
Fluo
resc
ence
Fig. S11 Digestion time-dependent fluorescence change of the sensing system.
[F-pEcoR] = 100 nM. Reaction temperature was 37 °C.
Electronic Supplementary Material (ESI) for AnalystThis journal is © The Royal Society of Chemistry 2013
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
Electronic Supplementary Material (ESI) for AnalystThis journal is © The Royal Society of Chemistry 2013
13. Optimization of L-GO concentration for Hind III detection
0 2 4 6 8 10 120
20
40
60
80
100
120
0 2 4 6 8 1 0 1 202468
1 01 2
F/F 0
Fluo
resc
ence
L-GO concentration (g/100 L)
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.
Electronic Supplementary Material (ESI) for AnalystThis journal is © The Royal Society of Chemistry 2013
14. Hind III activity detection using the proposed Hind III sensor.
500 520 540 560 580 600 6200
20
40
60
80
100
120
Fluo
resc
ence
(a)0.3 U/100 L
0
Wavelength (nm)
0 1 2 3 4 50
20
40
60
80
100
120
140
160
180
0.00 0.05 0.10 0.15 0.20 0.25 0.30
20
40
60
80
100
120
(b)
Fluo
rese
nce
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.
Electronic Supplementary Material (ESI) for AnalystThis journal is © The Royal Society of Chemistry 2013
/ColorImageDict > /JPEG2000ColorACSImageDict > /JPEG2000ColorImageDict > /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 150 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages false /GrayImageDownsampleType /Bicubic /GrayImageResolution 150 /GrayImageDepth 8 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /FlateEncode /AutoFilterGrayImages false /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict > /GrayImageDict > /JPEG2000GrayACSImageDict > /JPEG2000GrayImageDict > /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages false /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /FlateEncode /MonoImageDict > /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False
/CreateJDFFile false /Description >>> setdistillerparams> setpagedevice