Electronic Supplementary Material (ESI) for Chemical Communications.
This journal is © The Royal Society of Chemistry 2014
Supporting Information
Label-free, isothermal and ultrasensitive electrochemical
detection of DNA and DNA 3’-phosphatase by using a
cascade enzymatic cleavage strategy
Shufeng liu*, Tao Liu, and Li Wang
Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College
of Chemistry and Molecular Engineering, Qingdao University of Science and
Technology, Qingdao 266042, China. E-mail: [email protected]
Table of contents
Experimental section----------------------------------------------------------------------S2-S3
Table S1 ------------------------------------------------------------------------------------S4
Figure S1 -----------------------------------------------------------------------------------S5
Figure S2 -----------------------------------------------------------------------------------S6
Figure S3 -----------------------------------------------------------------------------------S7
Table S2 ------------------------------------------------------------------------------------S8
Figure S4------------------------------------------------------------------------------------S9
Figure S5------------------------------------------------------------------------------------S10
Figure S6------------------------------------------------------------------------------------S11
Figure S7------------------------------------------------------------------------------------S12
Figure S8------------------------------------------------------------------------------------S13
Figure S9------------------------------------------------------------------------------------S14
Figure S10----------------------------------------------------------------------------------S15
Figure S11----------------------------------------------------------------------------------S16
References----------------------------------------------------------------------------------S17
S1
Experimental section.
Chemicals and reagents. 6-Mercaptohexanol was purchased from Sigma-Aldrich (St.
Louis, MO, USA). The Bst DNA polymerase, Large Fragment, Lambda Exonuclease,
Nb.BbvCI nicking endonuclease, and T4 polynucleotide kinase phosphatase (PNKP)
were purchased from New England Biolabs Ltd. (Ipswich, MA, USA). Human
thrombin was supplied by Dingguo Biotech Co., Ltd. (Beijing, China). Lysozyme was
bought from Tiangen Biotech Co, Ltd. (Beijing, China). Bovine serum albumin
(BSA), human IgG, deoxyribonucleoside triphosphates (dNTPs) and fetal bovine
serum were purchased from Sangon Biotech. Co., Ltd. (Shanghai, China). All other
chemicals were of analytical grade and used as received. The HPLC-purified
oligonucleotide sequences are purchased from Sangon Biotech. Co., Ltd. (Shanghai,
China) and listed in Table S1.
Apparatus. All electrochemical measurements were carried out by using a CHI 660D
electrochemical workstation (CH Instruments, Shanghai, China) at room temperature.
A conventional three-electrode system was used, which comprised a gold working
electrode (2 mm diameter), a platinum wire auxiliary electrode, and a Ag/AgCl
reference electrode.
Electrode Pretreatment. The gold electrode was cleaned by immersion in a freshly
prepared piranha solution (a 3:1 v/v mixture of concentrated H2SO4 and 30% H2O2)
for 20 min, followed by a thoroughly rinse with ultrapure water. Then the electrode
was polished on a microcloth (Shanghai Chenhua Inc., China) with 50 nm alumina
slurry to obtain a mirror surface, followed by sonication in acetone and ultrapure
water for 5 min each, to remove residual alumina powder. The well-polished electrode
was then subjected to electrochemical pretreatment by cycling the potential between 0.2 and 1.5V in H2SO4 (0.5 M) at a scan rate of 100 mV s-1 until a stable cyclic
voltammogram was obtained, and then the cleaned electrode was allowed to be dried
at room temperature.
Immobilization of hairpin DNA probe (HP) on Au Surface. The assembly of
hairpin DNA probe (HP) on the electrode surface was carried out according to the
S2
following procedures. The HP1 is used for target DNA detection and HP2 or HP3 is
used for PNKP detection. The HP was firstly heated to 90 °C for 5 min and then
allowed to cool to room temperature for at least 2 hours before use. Then, the cleaned
gold electrode was incubated into 50 µL of 1 µM HP in 20 mM Tris-HCl buffer (0.1
M NaCl, 10 µM TCEP, pH 7.4) for 12hr at room temperature and then thoroughly
rinsed with ultrapure water and dried under a stream of nitrogen gas. The electrode
was subsequently immersed in 1 mM MCH solution for 1 hour to remove the
nonspecific DNA adsorption. Then the electrode surface was rinsed thoroughly and
dried in nitrogen.
Target DNA recognition and one-pot cascade enzymatic cleavage. The DNA
hybridization and enzymatic amplification experiments were performed with the
incubation of HP1 assemblied electrode into 50 µL 20mM Tris-HCl buffer (140mM
NaCl, 5mM MgCl2, pH 7.4) consisting 10 U nicking endonuclease, 7.5 U lambda
exonuclease, 8 U Bst DNA polymerase, 1µM primer, 200 µM dNTPs and various
concentration of target DNA. All reactions were performed at 37oC for 60 min (except
for the time-course study). Then, the electrode was thoroughly rinsed and dried in
nitrogen.
Assay for PNKP activity. The HP2 immobilized electrode was incubated into 50 µL
20mM Tris-HCl buffer (140mM NaCl, 5mM MgCl2, pH 7.4) containing different
concentration of PNKP at 37oC for 60 min. Then, the electrode was thoroughly rinsed
and incubated into 50 µL 20mM Tris-HCl buffer (140mM NaCl, 5mM MgCl2, pH7.4)
consisting 10 U nicking endonuclease, 7.5 U lambda exonuclease, 8 U Bst DNA
polymerase, 200 µM dNTPs for 60 min at 37oC. After the reaction, the electrode was
rinsed and dried in nitrogen.
Electrochemical detection. The above treated gold electrode, as the working
electrode, was immersed into the electrochemical cell containing 5 mM
[Fe(CN)6]3−/4−and 1 M KNO3 for electrochemical measurement. Before measurements,
the electrolyte solution should be thoroughly purged with high purity nitrogen for
about 20 min. The differential pulse voltammograms (DPV) were recorded with the
potential window from 0.6 V to -0.1 V. The electrochemical impedance spectra (EIS)
S3
were recorded with the frequency range from 0.1 Hz to 10 kHz.
Table S1. DNA sequences used in the experimentsa
Name
Sequence (5’ to 3’)
HP1
SH-(CH2)6-
AAGCTGAGGTCTTGGACTAGATCTTCCAGTGTGAT
GAAGTCCAAGATTT
HP2
SH-(CH2)6-GCTGAGGAGAGATACGCACCTAAAAAGGGTGCG
TA-PO4
HP3
SH-(CH2)6-GCTGAGGAGAGATACGCACCTAAAAAGGGTGCG
TD
1MT
2MT
NC
TA-OH
TCATCACACTGGAAGACTC
TCATCTCACTGGAAGACTC
TCATCTCACGGGAAGACTC
ACGTTGCATATCGACTAGC
HP1 is used for target DNA detection. HP2 and HP3 are used for PNKP detection. TD, 1MT,
2MT and NC denote target DNA, single-base mismatched DNA, two-base mismatched DNA and
non-complementary DNA, respectively. The underlined letters in 1MT and 2MT indicate the
mismatched bases.
a
S4
Figure S1. Optimization of probe DNA (HP1) immobilization concentration. The
used concentrations are 0.1, 0.5, 1.0, 1.5, 2.0 µM, respectively. The target DNA
concentration is 0.1 nM. The peak current increase for y-axis indicates the
electrochemical response difference in the presence and absence of target DNA. The
error bars represent the standard deviation of three measurements.
S5
Figure S2. (A) The effect of the nicking endonuclease concentration on the
electrochemical response of the sensing system for target DNA detection. The studied
amounts were 4, 6, 8, 10 and 12 U. (B) Optimization of lambda exonuclease amount
for target DNA detection. A series of amounts including 3, 4.5, 6, 7.5 and 9 U were
used. The concentration of used target DNA was 0.1 nM. The error bars represent the
standard
deviation
of
S6
three
measurements.
Figure S3. (A) The time response of DPV peak current toward target DNA (0.1 nM)
detection in the presence of polymerase, lambda exonuclease and nicking
endonuclease (square), and absence of lambda exonuclease (triangle). (B) The
optimization of reaction temperature for target DNA (0.1 nM) detection. The current
increase for y-axis indicates the electrochemical response difference in the presence
and absence of target DNA. The error bars represent the standard deviation of three
measurements.
S7
Table S2. Comparison of detection performance for current fabricated DNA
biosensor with the reported electrochemical methods
Method
Detection limit
strategy
Signal reporter
Ref.
ACV
2×10-9 M
Lambda exonuclease based target
Methylene blue
1
SWV
2×10−14
RuHex
2
DPV
1.67×10-13 M
Ferrocence
3
EIS
4.2×10−11 M
Fe(CN)63-/4-
4
DPV
1×10−15 M
RuHex
5
Fe(CN)63-/4-
6
Circular strand-displacement
Alkaline
7
polymerase reaction and HCR
phosphatase
Restriction endonuclease-aided
Methylene blue
8
Methylene blue
9
Fe(CN)63-/4-
This work
recyling
M
Exonuclease III-aided target
recycling
Nicking endonuclease assisted
signal amplification
Lambda exonuclease based target
recycling
Exonuclease I cleavage and
biobarcode amplification
EIS
1×10−14 M
Exo III-based target recycling and
graphene/Au nanocomposites
×10−14
DPV
0.8
M
DPV
1×10-9 M
ACV
1.4×10−11
DPV
1×10−14 M
target recycling
M
Restriction endonuclease-aided
target recycling
Cascade enzymatic cleavage
amplification
Alternating-current voltammogram (ACV); Square wave voltammogram (SWV); Differential
pulse voltammogram (DPV); Electrochemical impedance spectroscopy (EIS)
S8
Figure S4. (A) DPV responses toward the blank and four various DNA sequences
including complementary target DNA (TD), single-base mismatched DNA (1MT),
two-base mismatched DNA (2MT), and non-complementary DNA (NC). The
concentrations of various DNA sequences were all 0.1 nM. (B) The bar chart of the
DPV responses toward various DNA sequences. Error bars represent standard
deviations of measurements (n=3).
S9
Figure S5. (A) The schematic illustration for target DNA detection in the absence of
lambda exonuclease. It could be denoted as a one-recyling amplification strategy. (B)
DPV responses of the fabricated one-recycling amplification system toward the
analysis of different concentrations of target DNA: (a) 0 pM, (b) 1 pM, (c) 10 pM, (d)
20 pM, (e) 50 pM, (f) 100 pM, (g) 1 nM, (h) 10 nM. (C) The linear plot of DPV peak
currents vs target DNA concentration from 1 pM to 100 pM. The error bars
represented the standard deviation of three repetitive measurements.
S10
Figure S6. DPV peak current obtained for the fabricated DNA biosensor in buffer and
2% fetal bovine serum (50 fold diluted) spiked with two different target DNA
concentrations. The error bars represent the standard deviation of three measurements.
S11
Figure S7. (A) Cyclic voltammetric characterization; (B) Differential pulse
voltammetric
characterization;
(C)
Electrochemical
impedance
spectroscopic
characterization. The curves (a), (b), (c) and (d) in A, B and C were obtained for bare
gold electrode, HP2 modified electrode, PNKP treated electrode and the control
electrode in the absence of PNKP, respectively. The used PNKP concentration is 1 U
mL-1.
S12
Figure S8. (A) The corresponding DPV responses for HP2 assembled electrode after
3’-dephosphorylation reaction (black line), and HP3 assembled electrode in the
absence of PNKP (red line), respectively. The used PNKP concentration is 1 U mL-1.
(B) Bar chart for DPV responses obtained at different experimental conditions toward
PNKP detection. The x-axis shows the corresponding experimental conditions. The
error bars represented the standard deviation of three repetitive measurements.
S13
Figure S9. Time optimization of dephosphorylation reaction. The used time were 15,
30, 45, 60, 90 min, respectively. The error bars represent the standard deviation of
three measurements. The inset shows the corresponding DPV responses at different
time.
S14
Figure S10. (A) Schematic illustration for the detection of DNA 3’-phosphatase in
the absence of lambda exonuclease. (B) The bar chart of DPV responses obtained in
the presence and absence of lambda exonuclease toward the detection of different
concentrations of PNKP. The error bars represented the standard deviation of three
repetitive measurements.
S15
Figure S11. Selectivity of the fabricated sensor towards PNKP compared to other
interfering proteins. The concentration of PNKP is 1 U mL-1. The concentration for
other interfering proteins like BSA, thrombin, lysozyme and IgG are all 0.1 μM. The
“Blank” at the X-axis indicate the electrochemical response in the absence of PNKP.
S16
References
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