Author’s Accepted Manuscript
A novel fluorescent sensor for mutational p53 DNAsequence detection based on click chemistry
Suyan Qiu, Xianghui Li, Wenming Xiong, Lidan Xie,Longhua Guo, Zhenyu Lin, Bin Qiu, Guonan Chen
PII: S0956-5663(12)00606-9DOI: http://dx.doi.org/10.1016/j.bios.2012.08.065Reference: BIOS5396
To appear in: Biosensors and Bioelectronics
Received date: 24 August 2012Accepted date: 31 August 2012
Cite this article as: Suyan Qiu, Xianghui Li, Wenming Xiong, Lidan Xie, Longhua Guo,Zhenyu Lin, Bin Qiu and Guonan Chen, A novel fluorescent sensor for mutational p53DNA sequence detection based on click chemistry, Biosensors and Bioelectronics, http://dx.doi.org/10.1016/j.bios.2012.08.065
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1
A novel fluorescent sensor for mutational p53 DNA sequence 1
detection based on click chemistry 2
3
Suyan Qiu, Xianghui Li, Wenming Xiong, Lidan Xie, Longhua Guo, Zhenyu Lin,∗ Bin 4
Qiu, Guonan Chen∗ 5
MOE Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key 6
Laboratory of Analysis and Detection Technology for Food Safety, Department of 7
Chemistry, Fuzhou University, Fuzhou, Fujian, 350002, China 8
9
Abstract 10
A novel fluorescent sensor for DNA sequence has been designed by taking advantages 11
of copper nanoparticles (CuNPs) selectively formed on double stranded (ds) DNA 12
template and Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Copper(II) 13
is derived from CuNPs which previously formed on the dsDNA template, and then 14
copper(II) is reduced to copper(I) by ascorbate, which in turn induced CuAAC reaction 15
between the weak-fluorescent compound (3-azido-7-hydroxycoumarin) and propargyl 16
alcohol to form a strong fluorescence compounds (1,2,3-triazole compounds). Since 17
CuNPs are accumulated efficiently in the major groove of dsDNA and ssDNA has no 18
groove, indicate that the proposed sensor owns the merits of low detection limit, high 19
sensitivity and selectivity for mutational p53 sequence detection. Additionally, the 20
∗ Corresponding author; e-mail: [email protected] (G. Chen); [email protected] (Z. Lin); Fax: 86-591-22866135
2
method has been successfully applied to recognize the sequence which containing a 21
single-base mismatch in the short human p53 gene fragment. Furthermore, it has also 22
been applied to detect DNA sequence in complex medium (hela cellular homogenate) 23
with satisfactory results. 24
Keyword: fluorescent sensor; p53 sequence; copper nanoparticles; click chemistry; 25
single-nucleotide mismatch. 26
27
1. Introduction 28
Previous reports showed that copper nanoparticles (CuNPs) can be selectively formed 29
on the double stranded DNA (dsDNA) template, whereas not on the single stranded DNA 30
(ssDNA) template (Rotaru et al., 2010). The reason lies in that nanoparticles are 31
accumulated in the major groove of dsDNA which is absent in the ssDNA. And the 32
formation of CuNPs is quite efficient even at very low concentration of dsDNA template. 33
This characteristic has been applied to develop a label-free aptamer based sensor for 34
adenosine triphosphate (ATP) (Zhou et al., 2011). 35
Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction (Lutz, 2007) , one of the 36
most extensively studied click chemistry reactions, owns the characters of admirable 37
reaction kinetics, high efficiency and yielding, good biocompatibility and low chemically 38
reactive to the surrounding biological milieu (Jewett and Bertozzi, 2010). CuAAC 39
reactions have played significant roles in various fields, such as polymer materials (Chen 40
et al., 2011), macromolecules (Camponovo et al., 2009), bioconjucations (El-Sagheer and 41
Brown, 2010; Peters et al., 2010) and functional nanomaterials (Mader et al., 2010). 42
Several sensitive and selective analytical methods based on this reaction have been 43
3
reported, which reveals that the occurrence of CuAAC reaction can be catalyzed 44
quantitatively by copper(I) species (Zhou et al., 2008; Garner and Janda, 2010). But till 45
now, mostly studies were focused on the detection of copper(II) and ascorbic acid 46
species(Qiu et al., 2011; Qiu et al., 2011). The possibility of the application of CuAAC 47
reaction in other species detection has rarely been reported (Qu et al., 2011). 48
Mutations and deletions of p53 gene relate to human cancers or tumors frequently. 49
More than 50% of human cancers can be attributed to the mutations of p53 gene, which 50
may lead to the loss of transcriptional activation potency and the ability to bind DNA. 51
p53 mutations can occur at different sites among various cancers (Koshland, 1993; 52
Hollstein et al., 1996). For example, the CpG dinucleotide of codon 248 (CGG), one of 53
the strongest hot-spot of p53 gene, is frequently found their mutations in colon cancers 54
(Mancuso et al., 1997). And previous studies indicated that the G to T transversion at the 55
third base pair position of codon 249 (AGG) within p53 gene is usually discovered in 56
hepatocellular carcinoma (HCC) (Hsu et al., 1991; Coursaget et al., 1993). Therefore, the 57
detection of p53 mutational sequence is crucial in early cancer diagnosis and treatment. 58
In this study, a novel fluorescent sensor for DNA mutational sequence has been 59
presented through combining the advantages of CuNPs formed on the dsDNA template 60
and CuAAC reaction. The determination of the p53 DNA sequence containing a mutated 61
allele of codon 249 has been chosen as an example. Herein, CuNPs formed on the dsDNA 62
template has been oxidized to copper(II) by HNO3, and then, copper(II) is reduced to 63
copper(I) by ascorbate, which in turn induces the CuAAC reaction between the weak 64
fluorescent 3-azido-7-hydroxycoumarin and propargyl alcohol, and forms a strong 65
fluorescent compounds (1,2,3-triazole compounds) (Beatty et al., 2006; Droumaguet et 66
4
al., 2010). Due to the formation of CuNPs is highly selective to dsDNA template, so the 67
proposed method shows high specificity. And it has been applied to detect the p53 68
mutational sequence in complex medium (hela cellular homogenate). 69
70
2. Experimental Section 71
2.1 Chemicals 72
Sodium ascorbate, propargyl alcohol, 2,4-dihydroxy benzaldehyde, N-acetylglycine, 73
anhydrous sodium acetate, acetic anhydride, sodium azide and other reagents were 74
purchased from Alfa Aesar China (Tianjin) Co. Ltd. and used as received. 3-(N-75
Morpholino)-propane sulfonic Acid (MOPS) was obtained from Shanghai Chemical 76
Reagent Company (Shanghai, China). Streptavidin Magnespheres Paramagnetic Particles 77
(PMPs) was bought from Promega Corporation (Madison, USA). Copper sulfate 78
pentahydrate (CuSO4·5H2O) was isolated by crystallization of anhydrous copper sulfate 79
from water. Without special indication, all reagents and solvents were of analytical grade 80
or better and used without further purification. 81
Oligonucleotides DNA were synthesized by Shanghai Sangon Biotechnology Co. Ltd. 82
(Shanghai, China) and these sequences were listed in Table 1. The target sequence (DNA 83
2), from the codon 245 to 252 of the wild type p53 gene containing a single-nucleotide 84
mutation at codon 249, was fully complementary sequence of the probe DNA (DNA 1). 85
Four nucleotide mismatch sequences were used as targets for determining the selectivity 86
(mismatch bases are underlined): the sequence DNA 3 was the high frequency of 87
mutations at codon 248 of p53 sequence. The sequence DNA 4 was from the codon 245 88
5
to 252 of the wild type p53 gene, and DNA 5 was fully non-complementary sequence of 89
DNA 1. 90
2.2 Instruments 91
Fluorescence spectra were recorded on a Varian Cary Eclipse. Transmission electron 92
microscopy (Tecnai 20, FEI-Philips) were used to obtain high-resolution images with a 93
field emission gun operating at 200 kV. Mass spectra were detected using ion trap mass 94
spectrometry, while IR spectra were measured on a Nicolet 6700 FT-IR spectrometer. 95
UV-Visible absorption spectra were recorded using a Lambda 750 UV-Vis 96
spectrophotometer. 97
2.3 Synthesis of 3-azide-7-hydroxycoumarin 98
3-azide-7-hydroxycoumarin was synthesized according to the literature (Sivakumar et 99
al., 2004). 2,4-dihydroxy benzaldehyde (1.38 g, 10 mmol), N-acetylglycine (1.12 g, 10 100
mmol), anhydrous sodium acetate (30 mmol) were added into acetic anhydride (50 mL), 101
and then refluxed under stirring for 4 hours. The reaction mixture was poured onto ice to 102
obtain a yellow precipitate. After filtration, the yellow solid was washed by ice water, and 103
then refluxed in a solution containing HCl and ethanol (2:1, 30 mL) for 1 hour. Then, the 104
above solution was diluted by ice water (20 mL) and cooled in an ice bath. After NaNO2 105
(20 mmol) was added and stirred for 10 minutes, NaN3 (30 mmol) was added and stirred 106
for another 20 minutes. The filtered precipitate was produced and a brown solid was 107
obtained after drying under reduced pressure (0.86 g, 42.3%). 1H NMR (DMSO, 400 108
MHz) δ 6.76 (d, J = 2.13 Hz, 1 H), 6.80 (dd, J = 8.47 Hz, J = 2.28 Hz, 1 H), 7.48 (d, J = 109
8.54 Hz, 1 H), 7.61 (s, 1 H), 5.23 (s, 1 H). IR (KBr, cm-1): 3159 (s), 2117 (vs), 1688 (s), 110
6
1617 (m), 1310 (s). MS (EIS) m/z: calculated for C9H5N3O3: 203.0, found 202.5 ([M]-); 111
238.1 ([M + Cl] -). 112
2.4 Preparation of the fluorescent sensor for DNA assay 113
DNA probe and target DNA were dissolved in MOPS buffer solution (10 mM, pH 7.5) 114
consisting of NaCl (150 mM), MgCl2 (1.0 mM) and sodium ascorbate (1.0 mM). The 115
above solution was heated to 90 oC for 10 minutes and slowly cooled down to 60 oC; Then 116
incubated for 30 minutes to ensure the complete hybridization of the nucleic acids. After 117
this, added CuSO4 (50 µM) into the solution and incubated for 20 minutes at 60 oC; and 118
then rapidly cooled to room temperature again. Then 10 µL PMPs were added and taken 119
out by magnet after another 30 minutes of incubation period, followed by rinsing with 120
deionized water for three times. Then, 20 µL HNO3 (1.0 mM) was added and kept for 10 121
minutes, and then remove PMPs by magnet. Finally, a mixed MOPS buffer solution (10 122
mM, pH 7.5), including 3-azido-7-hydroxycoumarin (0.025 mM), propargyl alcohol 123
(0.025 mM) and sodium ascorbate (3.0 mM), was added into the above solution and 124
shaked for 3 hours in order to form fluorescent 1,2,3-triazole compounds by the CuAAC 125
reaction. The data of fluorescence emission spectra were collected at the excitation 126
wavelength of 395 nm at room temperature. Here, the fluorescence increase factor was 127
defined as (Fds-Fss)/Fss, where Fss and Fds were the fluorescence intensity of the sensing 128
system based on the formation of CuNPs on ssDNA template and dsDNA template, 129
respectively. All measurements were repeated three times and the standard deviation was 130
calculated as the error analysis. 131
132
133
7
2.5 Preparation and DNA assay in cellular homogenate 134
The cellular homogenate had been prepared according to the literature (Kong etal., 135
2011): firstly, the Hela cells (8.0 × 105 cells) were centrifuged for 5 min at 25 � (1000 136
rpm) and the supernatant was removed, and the cells precipitate was redispersed in 1.0 137
mL MOPS buffer solution (pH 7.5). Then, the above solution was subjected to a 138
sonication treatment for 30 minutes (with 4 seconds on and 8 seconds off) in an ice-water 139
bath using a probe-type sonicator (200 W). Finally, the cellular homogenate was stored at 140
4 °C. 141
The target DNA sequences (DNA 2, DNA 3 and DNA 4) were added into the diluted 142
cellular homogenate samples (1.0×105 cells/mL), respectively, and then DNA probe 143
(DNA 1) was added also. According to the procedure of the section 2.4, DNA sequences 144
were detected in cellular homogenate. The data of fluorescence emission spectra were 145
collected at the excitation wavelength of 395 nm at room temperature. 146
147
3. Results and Discussion 148
Principle of the fluorescent sensor for DNA sequence determination 149
Several sensors have been reported based on the fluorescence of CuNPs (Zhou et al., 150
2011). However, these methods exist certain drawbacks. First, the fluorescence of CuNPs 151
is very unstable. The reason may lie in that CuNPs are readily to aggregate and easily 152
oxidized to CuO at atmosphere, which greatly influence their fluorescence (Darugar et 153
al., 2006). Second, these methods need harsh conditions, such as inert atmosphere in full 154
process. To overcome this drawback, a new strategy has been proposed (shown in Fig. 1). 155
CuNPs are formed on the dsDNA template firstly, and then enriched by the strong affinity 156
8
interaction between the biotin and streptavidin magnespheres paramagnetic particles 157
(PMPs), isolated by magnet and rinsed three times with deionized water. Through 158
oxidization by HNO3, copper(II) can be produced (Mao et al., 2007) and which is reduced 159
to copper(I) by sodium ascorbate, and copper(I) in turn catalyzes CuAAC reaction 160
between the weak-fluorescent 3-azido-7-hydroxycoumarin and propargyl alcohol, and 161
forms a fluorescent 1,2,3-triazole compounds. By this means, the effect of CuNPs 162
fluorescence can be avoided and no harsh conditions needed. CuNPs can be selectively 163
accumulated in the major groove of dsDNA which is absent in the ssDNA, so ssDNA can 164
not act as an efficient template for the CuNPs formation, no CuAAC reaction between 3-165
azido-7-hydroxycoumarin and propargyl alcohol occurs, thus no 1,2,3-triazole 166
compounds formed. Based on these, the proposed method can be applied to discriminate 167
DNA sequence with high selectivity. 168
The preferred position for Fig. 1 169
3.2 Feasibility of the sensor 170
Fig. 2A showed the TEM images of CuNPs formed on the ssDNA template and dsDNA 171
template, respectively. No CuNPs on the ssDNA template had been detected (inset in Fig. 172
2A), whereas several cubic CuNPs could be clearly observed on the dsDNA template, and 173
the diameters of the CuNPs were about 65 nm. These results confirmed that dsDNA could 174
be acted as an efficient template for the formation of CuNPs. 175
The preferred position for Fig. 2 176
The fluorescence emission spectra and the color of the sensing system were depicted in 177
Fig. 2B. A weak fluorescence was detected in the system which contained no DNA or 178
ssDNA template (see curve a and b in Fig. 2B), and the corresponding colors under UV 179
9
lamp excitation (365 nm) were dark (see a and b in the inset of Fig. 2B). However, a 180
strong fluorescence could be detected in the presence of dsDNA template (see curve c in 181
Fig. 2B), and the corresponding color of the system was bright (see c in the inset of Fig. 182
2B). The reason for this phenomenon lied in that CuNPs were accumulated in the major 183
groove of the dsDNA, and then were reduced to copper(I) species, which in turn 184
catalyzed CuAAC reaction between 3-azido-7-hydroxycoumarin and propargyl alcohol to 185
form the fluorescent 1,2,3-triazole compounds, resulted in the increase of fluorescence 186
intensity, and the emission wavelength shifted from 463 nm to 471 nm. 187
188
3.3 Optimization of the sensing system 189
The effect of Cu(II) concentration had been studied first and shown in Fig. 3A. The 190
fluorescence increase factor (defined as (Fds-Fss)/Fss, where Fss and Fds were the 191
fluorescence intensity of the sensing system based on the formation of CuNPs on ssDNA 192
template and dsDNA template, respectively.) increased sharply with the enhancement of 193
Cu(II) concentration in the range of 5.0 ~ 50 µM, and then increase little over 50 µM. so 194
50 µM of Cu(II) was used in the subsequent experiments. 195
The relationship between the fluorescence increase factor and the CuAAC reaction 196
time of 3-azido-7-hydroxycoumarin and propargyl alcohol had been studied also (Fig. 197
3B). The time dependent curve of the fluorescence increase factor reached a saturation 198
plateau after 3 hours. Thus, 3 hours had been chosen as the best reaction time in the 199
subsequent studies. 200
The preferred position for Fig. 3 201
202
10
203
3.4 Target DNA sequence determination 204
Under optimal conditions, the fluorescence spectra at different target DNA sequence 205
concentrations had been detected and shown in Fig. 4A. The fluorescence intensity of the 206
sensing system increased gradually upon enhancement of the target DNA concentration, 207
when the concentration of target DNA was higher than 200 nM, the fluorescence intensity 208
has no significant increase (Fig. 4B). The reason for this phenomenon lied in that more 209
CuNPs were formed with increase of the target DNA concentration, more copper(I) 210
species was produced, and more fluorescent 1,2,3-triazole compounds were yielded 211
through the CuAAC reaction. When the target DNA concentration was higher than 200 212
nM, the CuAAC reaction went thorough, and the fluorescence intensity did not increase 213
further. In addition, there has a good linear relationship between the values of (Fds-Fss)/Fss 214
and the logarithm of target DNA concentrations in the range of 0.1 to 200 nM (see the 215
inset in Fig. 4B). The limit of detection for target DNA was calculated to be 52 pM 216
(S/N=3), which was lower than that of the early reported fluorescent methods (0.41 nM) 217
(Wang et al., 2008). The lower detection limit of the proposed method maybe attributed 218
to three reasons: i) the formation of CuNPs maybe sensitive to the dsDNA template; ii) 219
the copper(I) catalyzed azide-alkyne reaction allows highly sensitive to copper(I) species; 220
iii) CuNPs are dissolved and functioned as a catalyst for the CuAAC reaction which 221
contributes to the formation of the fluorescent 1,2,3-triazole compounds, and amplifies 222
the evolution of fluorescent signal related to the different target DNA concentrations. 223
The preferred position for Fig. 4 224
225
11
3.5 Specificity and practicality of the sensor 226
Fig. 5 showed the changes of fluorescent signal resulting from the sensing system on 227
the different dsDNA templates. The fluorescence intensity of the system based on CuNPs 228
formed on the fully complementary DNA template (Fig. 5A d) was much higher than that 229
on the fully non-complementary DNA template (Fig. 5A a) and DNA template containing 230
a single-nucleotide mismatch sequence (Fig. 5A b, c). Since no melting temperature of 231
the fully non-complementary and single-nucleotide mismatch duplexes DNA was over 60 232
oC except the fully complementary duplexes of the same size (Marathias et al., 2000). If 233
the hybridization temperature was set at 60 oC, most of them would exist in a single 234
stranded, and could not act as an efficient template for the formation of CuNPs (Rotaru et 235
al., 2010), led to the fluorescences of these systems were weak. In addition, the similar 236
results were also observed from the corresponding fluorescence colors. The color on the 237
fully complementary DNA template (see d in the inset of Fig. 5A) was much brighter 238
than that on the DNA templates containing the nucleotide mismatch sequence (see a, b, c 239
in the inset of Fig. 5A). These results suggested that the proposed method could be 240
applied to discriminate the nucleotide mismatch in the short DNA sequence, and could be 241
successfully applied to identify the nucleotide mutation in the short human p53 sequence. 242
The preferred position for Fig. 5 243
To test the practicality of the proposed sensor, the determination of the mutated DNA 244
sequence was carried out in the tumor cellular homogenate. The DNA sequence (DNA 2, 245
DNA 3 and DNA 4) was added to the diluted cellular homogenate samples (1.0×105 246
cells/mL) respectively to avoid the interference from background of cellular homogenate. 247
The results were shown in Fig. 5B, the responses of the mutated DNA sequences in the 248
12
cellular homogenate were similar to that in the ordinary buffer solution. These results 249
revealed that this sensing system could be applied to detect the fully complementary 250
DNA sequence in complex media. 251
252
4. Conclusions 253
In summary, a novel, highly sensitive and selective fluorescent sensor for p53 254
mutational sequence determination was developed by taking advantages of CuAAC 255
reaction and CuNPs formed on the dsDNA template. It was found that CuNPs were 256
selectively formed on the dsDNA template. Furthermore, it was quite efficient even at 257
very low concentration of dsDNA, which led to a lower detection limit as compared with 258
that of the early reported fluorescent methods. The sensitivity and selectivity also met the 259
requirements for most of biological assay. The results demonstrated that the proposed 260
method could be applied to recognize the short DNA sequence with the nucleotide 261
mismatch. In addition, this method had also been used to detect the p53 mutational 262
sequence in complex medium (hela cellular homogenate) with satisfactory results. 263
Furthermore, due to the proposed sensor has high specificity to identify the fully 264
complementary DNA sequence. If we can design a fully complementary DNA sequence 265
for the oncogene of chronic myelogenous leukaemia, and the pathogenic gene of 266
cymbidium mosaic virus, the proposed sensor may distinguish them with satisfactory 267
results. Therefore, we anticipate that such types of assay will expand the application of 268
click chemistry in analytical chemistry fields, and has potential applications in 269
biochemistry and clinical fields such as clinical assay, early cancer diagnosis and 270
treatment. 271
13
272
Acknowledgements 273
This project was financially supported by the National Basic Research Program of 274
China (No.2010CB732403), NSFC (21175024, 41076059), SRF for ROCS, SEM 275
(LXKQ201102), the Program for New Century Excellent Talents in Fujian Province 276
University (JA10011), and the Program for Changjiang Scholars and Innovative Research 277
Team in University (No. IRT1116). 278
279
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References 280
Beatty, K. E., Liu, J. C., Xie, F., Dieterich, D. C., Schuman, E. M., Wang, Q., Tirrell, D. 281
A., 2006. Angew. Chem. Int. Ed. 45, 7364-7367. 282
Camponovo, J., Ruiz,J., Cloutet, E., Astruc, D., 2009. Chem. Eur. J., 15, 2990-3002. 283
Chen, J., Liu, M., Chen, C., Gong, H., Gao, C., 2011. ACS Appl. Mat. Interfaces, 3, 3215-284
3223. 285
Coursaget, P., Depril, N., Chabaud, M., Nandi, R., Mayelo, V., LeCann, P., Yvonnet, B., 286
1993. Br. J. Cancer, 67, 1395-1397. 287
Darugar, Q., Qian, W., El-Sayed, M. A., 2006. J. Phys. Chem. B, 110, 143-149. 288
Droumaguet, C. L., Wang, C., Wang, Q., 2010. Chem. Soc. Rev. 39, 1233-1239. 289
El-Sagheer, A. H., Brown, T., 2010. Chem. Soc. Rev. 39, 1388-1405. 290
Garner, A. L., Janda, K. D., 2010. Angew. Chem. Int. Ed. 49, 9630-9634. 291
Hollstein, M., Shomer, B., Greenblatt, M., Soussi, T., Hovig, E., Montesano, R., Harris, C. 292
C., 1996. Nucleic Acids Res. 24, 141-146. 293
Hsu, I. C., Metcalf, R. A., Sun, T., Welsh, J. A., Wang, N. J., Harris, C. C., 1991 Nature, 294
350, 427-428. 295
Jewett, J. C., Bertozzi, C. R., 2010. Chem. Soc. Rev. 39, 1272-1279. 296
Kong, R. M., Zhang, X. B., Chen, Z., Meng, H. M., Song, Z. L., Tan, W., Shen, G. L., Yu, 297
R. Q., 2011. Anal. Chem. 83, 7603-7607. 298
Koshland, D. E., 1993. Science, 262, 1953. 299
Lutz, J. F., 2007. Angew. Chem. Int. Ed. 46, 1018-1025. 300
Mader, H. S., Link, M., Achatz, D. E., Uhlmann, K., Li, X., Wolfbeis, O. S., 2010. Chem. 301
Eur. J. 16, 5416-5424. 302
15
Mancuso, T., Aguilar, F., Pescarolo, M. P., Clerico, L., Russo, P., Parodi, S., 1997. 303
Nucleic Acids Res. 25, 3643-3648. 304
Mao, X., Jiang, J., Luo, Y., Shen, G., Yu, R., 2007. Talanta. 73, 420-424. 305
Marathias, V. M., Jerkovic, B., Arthanari, H., Bolton, P. H., 2000. Biochemistry. 39, 153-306
160. 307
Peters, W., Willnow, S., Duisken, M., Kleine, H., Macherey, T., Duncan, K. E., Litchfield, 308
D. W., Lüscher, B., Weinhold, E., 2010. Angew. Chem. Int. Ed. 49, 5170-5173. 309
Qiu, S., Gao, S., Liu, Q., Lin, Z., Qiu, B., Chen,G., 2011. Biosens. Bioelectron. 26, 4326-310
4330. 311
Qiu, S., Gao, S., Zhu, X., Lin, Z., Qiu, B., Chen, G., 2011. The Analyst, 136, 1580-1585. 312
Qu, W., Liu, Y., Liu, D., Wang, Z., Jiang, X., 2011. Angew. Chem. Int. Ed. 50, 3442-3445. 313
Rotaru, A., Dutta, S., Jentzsch, E., Gothelf, K., Mokhir, A., 2010. Angew. Chem. Int. Ed. 314
49, 5665-5667. 315
Sivakumar, K., Xie, F., Cash, B. M., Long, S., Barnhill, H. N., Wang, Q., 2004. Org. Lett. 316
6, 4603-4606. 317
Wang, M., Zhang, D., Zhang, G., Tang, Y., Wang, S., Zhu, D., 2008. Anal. Chem. 80, 318
6443-6448. 319
Zhou, Y., Wang, S., Zhang, K., Jiang, X., 2008. Angew. Chem. Int. Ed. 47, 7454-7456. 320
Zhou, Z., Du, Y., Dong, S., 2011. Anal. Chem. 83, 5122-5127. 321
322
323
16
Captions 324
Fig. 1 Scheme of the fluorescent sensor for DNA sequence determination based on 325
CuAAC reaction. 326
Fig. 2 (A) TEM images of CuNPs formed on a dsDNA template (DNA 1/ DNA 2) and 327
ssDNA template (DNA 1) (inset in Fig. 2A). (B) Fluorescence spectra after CuAAC 328
reaction based on CuNPs formed in the absence of DNA (a) and presence of ssDNA 329
(DNA 1) (b) or dsDNA template (DNA 1/ DNA 2) (c). Inset is the corresponding 330
photograph of fluorescence emission under UV lamp excitation (365 nm). Conditions: 331
DNA 1: 0.2 µM, DNA 2: 0.08 µM, CuSO4: 50 µM, 3-azido-7-hydroxycoumarin: 0.025 332
mM. 333
Fig. 3 Effects of Cu(II) concentrations (A) and CuAAC reaction time (B) on the 334
fluorescence increase factors [(Fds-Fss)/Fss]. Conditions: DNA 0.2 µM, MOPS buffer (10 335
mM, pH 7.5), 3-azido-7-hydroxycoumarin 0.025 mM. 336
Fig. 4 (A) Emission spectra of the sensing system based on CuNPs formed on the 337
different target DNA concentrations. From a to k : 0.0 nM, 0.1 nM, 0.5 nM, 1.0 nM, 3.0 338
nM, 6.0 nM, 10 nM, 40 nM, 80 nM, 200 nM and 500 nM, respectively. (B) The variations 339
of the fluorescence increase factors [(Fds-Fss)/Fss] with the different target DNA 340
concentrations. Inset: the calibration curve between the value of [(Fds-Fss)/Fss] and 341
logarithm of DNA concentrations. 342
Fig. 5 (A) Emission spectra of 1,2,3-triazole compounds based on CuNPs formed on the 343
different dsDNA templates: (a) DNA 1/ DNA 5; (b) DNA 1/DNA 3; (c) DNA 1/DNA 4; 344
(d) DNA 1/DNA 2. Inset: The corresponding photograph of fluorescence emission under 345
UV lamp excitation (365 nm). (B) The determination of the mutated DNA sequence in 346
17
the cellular homogenate: (a) DNA 1/DNA 3; (b) DNA 1/DNA 4; (c) DNA 1/DNA 2. 347
Conditions: 3-azido-7-hydroxycoumarin 0.025 mM, DNA 0.2 µM, CuSO4 50 µM, MOPS 348
buffer (10 mM, pH 7.5). 349
350
351
352
18
Table 1 Sequences of oligonucleotides DNA used in this experiment. 353
Number Sequences of DNA oligonucleotides
probe sequence (DNA 1) 5'-biotin-GAT GGG ACT CCG GTT
CAT-3' 18-mer
target sequence (containing a mutated
allele of codon 249 DNA 2)
5'-GGC ATG AAC CGG AGT CCC
ATC CTC-3' 24-mer
mismatch sequence of codon 248
(DNA 3)
5'-GGC ATG AAC CGC AGT CCC
ATC CTC-3' 24-mer
wild type p53 sequence (DNA 4) 5'-GGC ATG AAC CGG AGG CCC
ATC CTC-3' 24-mer
negative control (DNA 5) 5'-GGC GAT CCG ATT GAC ATT
CGT CTC-3' 24-mer
354
355
356
357
358
359
360
361
362
363
364
19
Fig. 1 365
366
367
368
369
370
371
Cu2+
AscorbateCu2+
Ascorbate
Cu2+
Ascorbate
Cu2+
Ascorbate
HNO
32
HNO
322
11
N3
OH
+N3
OH
+NN N
OH
AscorbateAscorbate
N3O
N3
HO ON3
O
N3
HO O
CuNPsCuNPs
MagnetMagnet
PMPsPMPsBiotinBiotin
Target DNATarget DNAProbe DNAProbe DNA
20
Fig. 2 372
373
374
375
376
377
378
400 450 500 550 6000
200
400
600
Wavelength/(nm)
Fluo
resc
ence
inte
nsity
/(a.u
.)
a
c a b c
B
200 nm200 nm
A
200 200
21
Fig. 3 379
380
381
382
383
384
385
0 20 40 60 80 1000
1
2
3
(Fds
- Fss)/
F ss
CCu(II) / ( μM)
A
0 1 2 3 4 5 60
1
2
3
(Fds
- Fss)/
F ss
Time / h
B
22
Fig. 4 386
387
388
389
390
391
392
0 100 200 300 400 500
1
2
3
4
(Fds
-Fss
)/F s
s
[DNA]/nM
B
-1 0 1 2 30
1
2
3
4
(Fds
-Fss)/
F ss
Log[DNA]/nM
400 450 500 550 6000
200
400
600
800
Fl/(a
.u.)
Wavelength/nm
Ak
a
23
Fig. 5 393
394
395
396
397
398
B
400 450 500 550 6000
150
300
450
600
FI/(a
.u.)
Wavelength/(nm)
a
c
A a b d c
400 450 500 550 6000
200
400
600
800
d
Wavelength/(nm)
FI/(a
.u.)
a
24
highlights: 399
● A novel fluorescent sensor for DNA sequence determination is developed. 400
● This biosensor is designed by taking advantages of CuAAC reaction and CuNPs. 401
● This biosensor has been used for detection of p53 sequence. 402
● This is good example of using click chemistry in biosensor. 403
404
405