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: gnchen@fzu.edu.cn (G. Chen); zylin@fzu.edu.cn (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

14

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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