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A Target-Lighted dsDNA-Indicator for High-Performance Monitoring of Mercury Pollution and Its Antagonists Screening Zhihe Qing, Lixuan Zhu, Xiaoxuan Li, Sheng Yang, Zhen Zou, JingRu Guo, Zhong Cao, and Ronghua Yang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02858 • Publication Date (Web): 25 Sep 2017 Downloaded from http://pubs.acs.org on September 28, 2017

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Environmental Science & Technology

1

A Target-Lighted dsDNA-Indicator for High-Performance

2

Monitoring of Mercury Pollution and Its Antagonists

3

Screening

4

Zhihe Qing,a,b,§,* Lixuan Zhu,a,§ Xiaoxuan Li,a Sheng Yang,a,b Zhen Zou,a,b Jingru

5

Guo,a Zhong Cao,a Ronghua Yang,a,b,*

6

a

7

Transportation, Hunan Provincial Engineering Research Center for Food Processing

8

of Aquatic Biotic Resources, School of Chemistry and Biological Engineering,

9

Changsha University of Science and Technology, Changsha 410114, P. R. China.

Hunan Provincial Key Laboratory of Materials Protection for Electric Power and

10

b

11

Chemistry and Chemical Engineering, Molecular Science and Biomedicine

12

Laboratory, Hunan University, Changsha 410082, P. R. China.

13

§

14

*To whom correspondence should be addressed:

15

E-mail: [email protected]; [email protected]; Fax: +86-731-88822523.

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of

Z.Q. and L.Z. as the co-first authors contributed equally to this work.

16 17 18 19 20 21 22

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ACS Paragon Plus Environment


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ABSTRACT

24

As well known, the excessive discharge of heavy-metal mercury not only destroys

25

the ecological environment, bust also leads to severe damage of human health after

26

ingestion via drinking and bioaccumulation of food chains, and mercury ion (Hg2+) is

27

designated as one of most prevalent toxic metal ions

28

high-performance monitoring of mercury pollution is necessary. Functional nucleic

29

acids have been widely used as recognition probes in biochemical sensing. In this

30

work,

31

iodine)-9H-carbazol -9-yl)] butanoate (EBCB), has been synthesized and found as a

32

target-lighted DNA fluorescent indicator. As a proof-of-concept, Hg2+ detection was

33

carried out based on EBCB and Hg2+-mediated conformation transformation of a

34

designed DNA probe. By comparison with conventional nucleic acid indicators,

35

EBCB held excellent advantages, such as minimal background interference and

36

maximal sensitivity. Outstanding detection capabilities were displayed, especially

37

including simple operation (add-and-read manner), ultra-rapidity (30 s), and low

38

detection limit (0.82 nM). Furthermore, based on these advantages, the potential for

39

high-performance screening of mercury antagonists was also demonstrated by the

40

fluorescence change of EBCB. Therefore, we believe that this work is meaningful in

41

pollution monitoring, environment restoration and emergency treatment, and may

42

pave a way to apply EBCB as an ideal signal transducer for development of

43

high-performance sensing strategies.

a

carbazole

derivative,

in drinking water. Thus, the

ethyl-4-[3,6-bis(1-methyl-4-vinylpyridium

44

2


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INTRODUCTION

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By virtue of its excellent physicochemical properties, including programmable

47

coding, easy synthesis, functional stability, and outstanding recognition ability (e.g.

48

Watson-Crick

49

configuration matching), deoxyribonucleic acid (DNA) has been not only the carrier

50

of genetic information, but also an ideal candidate for constructing molecule probes,

51

such as molecular beacon (MB), DNAzyme and aptamer.[1-7] As well known, the

52

signal conversion of DNA probes is mainly based on the transformation between

53

different DNA conformations, and is always one of the concern focuses in

54

constructing DNA probes.[8-12]

base-pairing,

DNA–metal

base-pairing

and

target-induced

55

In the signal generation of DNA probe-based sensors, the fluorescent method has

56

attracted most attention because of its multiformity, fast analysis speed and high

57

sensitivity. One strategy is based on labeling technique of fluorescent dye. In this

58

strategy, dyes (fluorophore and/or quencher) are labeled at the specific sites of DNA

59

probes, and then the signal generation is accompanied with the recognition events

60

between targets and dye-labeled DNA probes. The labeling-based strategy is widely

61

used for biochemical analysis, but extensive design and modification are always

62

required to prepare probes, and time-consuming optimization is generally needed to

63

obtain ideal sensing performance. Another alternative strategy is dependent on

64

fluorescent intercalating indicators,[13-18] which can spontaneously bind nucleic acids,

65

as a label-free fluorescent strategy. Despite the simplicity and convenience of

66

indicator-based methods, several deficiencies are always involved, such as high

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background interference and low sensitivity, which stem from the strong

68

self-fluorescence of indicators, and/or the nonspecific fluorescence enhancement

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induced by both ssDNA and dsDNA, lacking absolute selectivity for a certain DNA

70

conformation. The improving of the sensing performance, in the nucleic acid

71

indicator-based sensing, is generally dependent on additional processing, such as

72

enzyme-catalyzed signal amplification and nanomaterials-mediated background

73

suppression.[19-22] Thus, it is of challenge and significance to synthesize and screen a

74

DNA

75

target-dependent lighting of fluorescence with

conformation-specific

indicator

for

biochemical

sensing,

achieving

minimal background interference.

76

In this work, we were inspired to synthesize and screen a target-lighted DNA

77

fluorescent indicator for highly-effective biochemical sensing. As a proof-of-concept,

78

mercury ion (Hg2+), which can result in severe damage of human health after

79

ingestion via drinking and bioaccumulation of food chains, and is the most poisonous

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heavy metal in drinking water,[23-25] was chosen as the model target, thymine-rich

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(T-rich) DNA was designed as the probe, thymine-Hg2+-thymine (T-Hg-T)

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base-paring was designed to mediate the probe transformation from fully-melted

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ssDNA to double-stranded or hairpin DNA.[26-28] As shown in the Figure 1, when

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conventional indicators were used to monitor the target-induced probe conformation

85

transformation, there was high background interference for target detection, because

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of their strong self-fluorescence or lacking of DNA conformation selectivity in

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fluorescence lighting; and additional processing (e.g. nanomaterials-mediated

88

background suppression) was needed to improve sensing performance. Attractively,

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when

90

iodine)-9H-carbazol -9-yl)] butanoate (EBCB) was synthesized and used to monitor

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the target-induced probe conformation transformation, little self-fluorescence of

92

EBCB and negligible effect from single-stranded probe was found, and strong

93

fluorescence signal was detected from target-mediated dsDNA, resulting in minimal

94

background interference and high sensitivity for target sensing. Because the lighting

95

of EBCB fluorescence was completely dependent on the addition of the target, it was

96

rationally named as a target-lighted DNA fluorescent indicator. By virtue of its

97

minimal background interference and high sensitivity, EBCB was further exploited

98

for the screening of mercury antagonists, which are desirable for emergency treatment

99

and environment restoration.

100

a

carbazole

derivative,


EXPERIMENTAL SECTION

101

Chemicals and Apparatus. All DNA sequences used in this work are listed in

102

Table S1 and were purchased in Sangon Biotech Co., Ltd. (Shanghai). The

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fluorescence

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iodine)-9H-carbazol-9-yl)] butanoate (EBCB) was synthesized following our previous

105

method (Figure S1).[29] Conventional nucleic acid indicators, gold view (GV), SYBR

106

gold (SG), SYBR green I (SGI), and ethidium bromide (EB) were purchased from

107

Dingguo Biotechnology CO., Ltd (Beijing, China). 3-(N-morpholino) propanesulfonic

108

acid (MOPS), Hg(NO3)2 and other inorganic salts were obtained from Sinopharm

109

Chemical Reagent Co., Ltd. (China). The MOPS buffer contained 10 mM MOPS, 150

110

mM NaNO3 and 1mM Mg(NO3)2 (pH 7.2). Solutions were prepared using ultrapure

indicator,


5



111

water which was produced by a Millipore purification system (18.2 MQ resistivity).

112

All pH measurements were carried out by a model 868 pH meter (Orion). The

113

fluorescence spectra were carried on fluorospectrophotometer systems (PTI ASOC-10,

114

Photo Technology International, Birmingham, NJ, USA).

115

Screening Target-Lighted DNA-Indicators. In order to screen an ideal

116

target-lighted DNA fluorescent indicator, various fluorescence dyes, including EBCB,

117

GV, SG, SGI and EB, were used to monitor the Hg2+-mediated conformation change

118

of thymine-rich DNA (T20). Typically, 250 nM T20 and 500 nM EBCB were mixed

119

in 500 µL MOPS buffer solution (pH 7.2), then 10 µM Hg2+ was added into the above

120

solution and incubated for 5 min. The signal-to-background ratio was determined by

121

measuring the fluorescence intensities of the mixtures of EBCB and DNA in the

122

absence and presence of Hg2+. Similarly, conventional nucleic acid indicators were

123

also investigated respectively, according to the method mentioned above. Their

124

fluorescence spectra were recorded on a PTI ASOC-10 Fluorescence System, with

125

their maximum excitation wavelength, 470 nm for EBCB, 490 nm for SG, 495 nm for

126

SGI, 495 nm for GV, 500 nm for EB.

127

Monitoring Mercury Pollution. A single-stranded DNA with thymine-rich tails

128

and the ideal target-lighted DNA fluorescent indicator, EBCB, were applied to direct

129

detection of mercury pollution. Detailedly, 100 nM EBVB and 100 nM probe were

130

added into a MOPS buffer of 500 µL, to form the EBCB/probe system. The

131

fluorescence titrations were carried out via gradually adding the stock solution of

132

Hg2+ in an incremental fashion. Other metal ions (10 equiv.), including Li+, K+, Ag+,

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CH3Hg+, Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Pb2+, Zn2+, Cu2+, Cd2+, Co2+, Ni2+, Sn2+, Cr3+,

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Al3+ and Fe3+, were used to investigate the selectivity of EBCB/probe toward the Hg2+

135

detection.

136

For the monitoring of mercury pollution in real samples, we collected two kinds of

137

real water including lake water and river water from Taozi Lake beside Yuelu

138

mountain and Xiangjiang River, respectively. The lake water and river water were

139

filtered by a 0.22-µm syringe filter to remove insoluble material. The analytes were

140

prepared by spiked real water samples without or with Hg2+ of different concentration

141

levels. The detection procedure was the same as that mentioned above，all detections

142

were repeated three times. The measured fluorescence intensities were used to assess

143

recovery rates and standard deviations corresponding to different samples. In addition,

144

for comparison, the samples were also detected by a classic method (inductively

145

coupled plasma mass spectrometry, ICP-MS), and the consistency between methods

146

were evaluated.

147

Screening Mercury Antagonists. To further explore the applications of our

148

proposed strategy, the screening of mercury antagonists was carried out. Several

149

model molecules were screened for their ability to chelate Hg2+ in aqueous solution.

150

Samples were prepared by adding 100 nM HP-4T and 100 nM EBCB in 500 µL

151

MOPS buffer solutions, followed by the addition of 1.0 µM Hg2+. Subsequently,

152

model molecules of different concentrations, including potassium iodide (KI),

153

magnesium nitrate (Mg(NO3)2), glucose, L-glutamic acid (Glu) and glutathione

154

(GSH), were introduced into above system for the reaction with Hg2+, respectively.

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155

Finally, the antagonism effect of each antagonist on Hg2+ could be directly determined

156

by the decay of fluorescence intensity. The spectra of EBCB were recorded in the

157

emission wavelength from 500 to 700 nm with excitation at 470 nm.

158

RESULTS AND DISCUSSION

159

Screening a Target-Lighted DNA Fluorescent Indicator. As well known,

160

intercalating indicators of nucleic acid have been intensely exploited and widely

161

applied in biochemical sensing in past years, and contributions from them to label-free

162

detection strategies have been made in some degree. However, it is unavoidable that

163

some limitations, such as high background interference and low sensitivity, are still

164

remained to be solved in sensing, because of strong self-fluorescence of conventional

165

indicators and lack of high specificity in discriminating nucleic acid conformations.

166

Improved methods to achieve high-performance sensing were always dependent on

167

additional processing, such as nanomaterial-mediated background suppression and

168

enzyme-catalyzed signal amplification. Thus, it is desirable to synthesize and screen a

169

DNA conformation-specific indicator, realizing target-dependent lighting of

170

fluorescence with low-background interference and high sensitivity.

171

Firstly, to realize target-dependent lighting of fluorescence, different DNA

172

fluorescent indicators were used to monitor the target-mediated DNA conformation

173

transformation. As a proof-of-concept, a poisonous and environment- contaminative

174

target, mercury ion (Hg2+), was chosen as the model target, thymine-Hg2+-thymine

175

(T-Hg-T) base-paring was designed to mediate the conformation transformation of a

176

thymine-rich DNA from fully-melted ssDNA (T20) to dsDNA (T20-Hg2+-T20). As 8


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shown in Figure 2A and B, conventional nucleic acid indicators either don’t response

178

to T20 and T20-Hg2+-T20 with strong self-fluorescence(e.g. goldview, GV), or

179

response to both T20 and T20-Hg2+-T20 without absolute specificity in discriminating

180

nucleic acid conformations(e.g. SYBR gold, SG). In these cases, it is not difficult to

181

find that there is obvious background interference for Hg2+ sensing, leading to low

182

sensitivity. Attractively, one can find that EBCB displays negligible self-fluorescence,

183

little effect on its fluorescence is observed from single-stranded T20, and high

184

fluorescence is observed in the presence of T20 and Hg2+ (Figure 2 C), indicating

185

low-background interference and high sensitivity for Hg2+ sensing. For direct

186

demonstration of detection performance, the signal-to-background ratio (F/ F0) was

187

introduced (Figure 2 D), where F0 was the fluorescence intensity of each indicator in

188

the presence of T20 but not Hg2+, F was that in the presence of T20 and Hg2+. It is

189

obvious that an outstanding signal-to-background ratio was displayed for Hg2+

190

sensing when EBCB was used as the signal reporter. Thus, EBCB has been screened

191

as an ideal target-lighted dsDNA fluorescent indicator, and holds great potential for

192

application in nucleic acid conformation transformation-mediated biochemical

193

sensing.

194

Subsequently, the mechanism of interaction between EBCB and double-stranded

195

T20-Hg2+-T20 was further investigated. From the UV-Vis absorption spectra of

196

EBCB/T20 system (Figure S2), one can see that there was obvious hypochromism and

197

redshift with the increase of Hg2+ concentration. This was because of the formation of

198

double-stranded T20-Hg2+-T20 with the addition of Hg2+ and the intercalating action

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of EBCB in the newly-formed T20-Hg2+-T20, in accordance with the fact that an

200

electronic interaction between the intercalator and dsDNA bases results in the

201

absorption hypochromism and redshift of the intercalator.30 Furthermore, the effect on

202

the fluorescence enhancement from the electrostatic interaction between the

203

negatively charged phosphate backbone of DNA and EBCB was also investigated.

204

With the gradually increase of sodium chloride (NaCl) concentration in the

205

EBCB/T20-Hg2+-T20 system, there is negligible change in fluorescence intensity

206

(Figure S3), indicating that the electrostatic interaction between the negatively

207

charged phosphate backbone of T20-Hg2+-T20 and EBCB has no effect on the

208

target-dependent

209

intercalating-dependent

210

T20-Hg2+-T20 maybe contribute to the high DNA-conformation specificity and high

211

sensitivity of Hg2+ detection.

fluorescence

enhancement

interaction

between

of

EBCB.

EBCB

Thus,

and

this

high

double-stranded

212

High-Performance Monitoring of Mercury Pollution. After the screening of

213

EBCB as an ideal target-lighted dsDNA fluorescent indicator, it was used for the

214

monitoring of Hg2+, and a single-stranded DNA with thymine-rich tails was designed

215

as the probe. When in the absence of Hg2+, the probe was in a fully-melted state, and

216

EBCB was non-fluorescent; after the addition of Hg2+, the probe was mediated into a

217

hairpin conformation with a stem of ten base-pairs and a loop of four cytosine

218

nucleotides, and EBCB would be lighted by the double-stranded stem of the hairpin

219

conformation (Figure 3A). A good feasibility was demonstrated for Hg2+ monitoring

220

with very low background interference (Figure 3B), and the signal response was very

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rapid with an enough detectable signal in several seconds (Figure 3C), when EBCB

222

was used as the signal reporter, and 30 s was chosen as the detection time (inset in

223

Figure 3C and Figure S4). In comparison, when conventional indicators were used

224

instead of EBCB, obvious interference from self-fluorescence and the effect of

225

single-stranded probe on dyes could be observed (Figure S5). Thus, a much higher

226

signal-to-background ratio was obtained by EBCB than other indicators.

227

To get better performance for Hg2+ detection by applying EBCB, some conditions

228

were optimized by the manner of signal-to-background ratio (F/F0). First, because the

229

regulation of counterweight between the stem and the loop of a hairpin probe is the

230

key to achieve high-performance detection, the probe DNA sequence with different

231

numbers of T-Hg-T base-pairs in the stem was optimized. As shown in Figures S3 and

232

Figures 3D, the sequence, HP-4T, which could form hairpin conformation with four

233

T–Hg2+–T base-pairs admixed in its stem, was selected as the optimal detection probe.

234

Then, buffer pH, operation temperature, and assistant magnesium ion (Mg2+)

235

concentration were optimized. As shown in Figure S7-9, pH 7.2 and 25 oC were

236

chosen as the optimal buffer pH and operation temperature, which is approximate to

237

ambient condition and convenient for detection operation; 1 mM Mg2+ in buffer was

238

optimized to assist Hg2+ detection.

239

Under optimal conditions, EBCB/HP-4T was used for Hg2+ detection. First,

240

EBCB/HP-4T fluorescence emission, responding to different concentrations of Hg2+,

241

was recorded. As shown in Figure 4A, with the increasing of Hg2+ concentration, the

242

fluorescence

emission

peak

of

EBCB

climbs

11


continually,

indicating


243

Hg2+-concentration-dependent transformation of probe conformation. The relationship

244

between signal-to-background ratio (F/F0) and Hg2+ concentration was plotted in

245

Figure S10, where F was the fluorescence intensity at 550 nm in the presence of Hg2+

246

of corresponding concentration, F0 was that of the blank. A good detection

247

performance was displayed, with a linear detection range from 2 to 120 nM and a

248

detection limit of 0.82 nM (inset in Figure S10). This high-sensitivity was due to the

249

target-lighted manner of EBCB, with little background interference. Then, the

250

selectivity for Hg2+ sensing was also investigated, as shown in Figure S11, a unique

251

emission peak of EBCB was induced by the introduction of Hg2+, and no response to

252

other ions; compared to the blank, a much higher signal-to-background ratio (F/F0)

253

was displayed for Hg2+, while little change was resulted in by other ions, and an

254

enough response could be detected after mixing other ions with Hg2+ (Figure 4B). In

255

addition, the selectivity was investigated when in the presence of competitive ions of

256

different concentrations (Figure S12), no interference was detected from competitive

257

ions even at a much higher concentration (1 mM). Thus, a high selectivity was

258

demonstrated for Hg2+ detection in this sensing strategy, this was because of the

259

specific coordination between thymine and Hg2+.[26,27] To directly display advantages

260

of this strategy, Hg2+ detection methods which were recently reported were

261

summarized in Table 1. The detection capability of our strategy not only meets the

262

requirement for Hg2+ detection in drinking water whose maximum level of Hg2+ is 10

263

nM permitted by US Environmental Protection Agency (EPA),[31,32] but also superior

264

to the developed methods, especially in simple operation, ultra-rapidity and low

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detection limit. 19,23,24,33-37

266

Finally, the practical application for monitoring mercury pollution was investigated,

267

different Hg2+-spiked water samples (including river and lake water) were

268

simultaneously detected by our approach and a classic method (inductively coupled

269

plasma mass spectrometry, ICP-MS), and the recovery percent and the consistency

270

between methods were calculated to evaluate the detection performance. As shown in

271

Table 2, high consistencies and satisfactory recoveries were achieved, indicating that

272

the our strategy was reliable for the monitoring of Hg2+ from real water samples,

273

especially significant for Hg2+ detection in drink water.

274

Screening Mercury Antagonists. As well known, the ecological environment can

275

be destroyed by excessive emission of heavy metal ions, and severe damage to human

276

health can be resulted in by over-ingestion of toxic ions. Thus, it is strongly necessary

277

to obtain potent antagonists of a toxic substance, for emergency treatment and

278

environment restoration.[25,38] Here, by virtue of its typical properties, including

279

minimal background interference, high sensitivity and rapid response, the proposed

280

strategy was further exploited to screen antagonists of Hg2+. If in the presence of an

281

antagonist in the detected water system, the antagonist would bind with Hg2+ to form

282

a stable complex, resulting in decreasement of dissociative Hg2+, disfavoring the

283

conformation transformation of the probe, and dropping off the fluorescence intensity

284

of EBCB. Thus, the antagonism effect can be evaluated by the fluorescence change.

285

As the proof-of-concept, several model molecules were used and screened,

286

including inorganic salt (KI and Mg(NO3)2), monosaccharide (glucose), amino acid

13



287

(aminoglutaric acid, Glu) and small peptide (glutathione, GSH). After incubation of

288

each model molecule with Hg2+ for 5 min, the probe HP-4T and EBCB were added

289

into the above system, and fluorescence was measured. As shown in Figure 5A, one

290

can see that there was little effect on the fluorescence from Mg(NO3)2, glucose and

291

Glu, while obvious decline of fluorescence intensity can be observed when KI and

292

GSH were used, indicating strong antagonism effect from KI and GSH against Hg2+.

293

Then, the concentration-dependent antagonism was also investigated (Figure S13 and

294

Figure 5B), with the concentration increasing of model molecules, negligible change

295

of fluorescence intensity could be detected when Glu were used, while significant

296

decreasement of fluorescence intensity could be observed when the antagonists KI and

297

GSH were used; and a stronger antagonism from KI was implied with a large slope of

298

descent than that from GSH. Therefore, these results demonstrated that this proposed

299

strategy not only can used for high-performance detection of Hg2+, but also can be

300

applied for the effective screening of mercury antagonists, due to its rapid response,

301

minimal background interference and high sensitivity.

302

ASSOCIATED CONTENT

303

Supporting Information

304

More experimental results and figures as noted in text, including the sequence

305

information of DNA used in this study, the synthetic route of EBCB, UV-Vis

306

absorption spectra, effect of ionic strength on fluorescence enhancement, the

307

optimization of conditions, et. al. This material is available free of charge via the

308

Internet at http://pubs.acs.org. 14


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AUTHOR INFORMATION

310

Corresponding Author

311 312

* E-mail: [email protected]; [email protected]. Author Contributions

313

§

314

Notes

315

316

Z.Q. and L.Z. as the co-first authors contributed equally to this work.

The authors declare no competing financial interest.

ACKNOWLEDGMENT

317

We are grateful for the financial support through the National Natural Science

318

Foundation of China (21605008, 21575018, 21505006), the Hunan Provincial Natural

319

Science Foundation (2016JJ3001), the Scientific Research Fund of Hunan Provincial

320

Education Department (16C0032, 16C0033), the Open Fund of State Key Laboratory

321

of Chemo/Biosensing and Chemometrics of Hunan University (2015003) and Hunan

322

Provincial Engineering Research Center for Food Processing of Aquatic Biotic

323

Resources (2016GCZX04).

324

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441 442 443

TOC/Abstract Art

444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459

21



460 461 462

463

Figures and Tables

464 (A) conventional fluorescent indicators background interference

◆

ssDNA

◆

◆

target (◆)

dsDNA

nanomaterials

target (◆)

background suppression (B) the target-lighted indicator, EBCB minimal background

◆

◆

465

◆

target (◆)

dsDNA

ssDNA

466

Figure 1. DNA conformation transformation-mediated sensing based on different

467

indicators. (A) Conventional indicators generally display high background

468

interference, and additional processing (e.g. nanomaterials-mediated background

469

suppression) was exploited to improve detection performance; (B) while EBCB was

470

here synthesized and found as a target-lighted DNA fluorescent indicator, with

471

minimal background interference and high sensitivity.

472 473 474 475 476 477

22


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Page 23 of 28


478 479 480 481

.6 .4 .2 550 600 650 Wavelength / nm

700

EBCB+T20+Hg EBCB+T20 EBCB

2+

.6

.2 0.0 500

550

600

650

700

Wavelength / nm

50 40 30

.4

20

.2

10 0

700

V

550 600 650 Wavelength / nm

G

0.0 500

.4

B

.8

.6

F / F0

Normalized fluorescence

1.0

.8

(D)

(C)

SG+T20+Hg2+ SG+T20 SG

C

0.0 500

1.0

EB

.8

GV+T20+Hg2+ GV+T20 GV

SG

1.0

(B) Normalized fluorescence


(A)

482 483

Figure 2. Monitoring the Hg2+-mediated T20 conformation transformation by

484

different DNA fluorescent indicators: (A) Gold View (GV), (B) SYBR Gold (SG), (C)

485

EBCB. (D) Direct demonstration of the monitoring capability by the manner of

486

signal-to-background ratio (F/ F0), where F0 was the fluorescence intensity of each

487

indicator in the presence of T20 but not Hg2+, F was that in the presence of T20 and

488

Hg2+.

489 490 491 492 493 494 23



(A)

Page 24 of 28

(B) EBVB+DNA+Hg2+ EBCB+DNA EBCB

Fluorescence

1e+6

Hg 2+ ( )

30 s

N

6e+5

N

4e+5

N

2e+5

550

650

700

(D)

12

1e+6

10 Hg2+ HP-4T EBCB buffer

-7 T

HP

HP

5T

4T

P-

P-

3T

250 500 750 1000

-6 T

0 H

0

4 2

620 640 660

0

6

H

4e+5

√

8

P-

6e+5

H

8e+5

F / F0

Fluorescence

600

Wavelength / nm

(C)

495

+

EtO

= probe with T-rich tails

2e+5

-

O

0

= EBCB

=

8e+5

Time / s

496

Figure 3. (A) Schematic representation for high-performance monitoring of mercury

497

pollution based on EBCB and T-Hg-T-mediated conformation transformation of DNA

498

probe with thymine-rich tails. (B) Fluorescence spectra of EBCB under different

499

conditions. The structure of EBCB is shown and the inset is the fluorescence image

500

(left: without Hg2+, right: with Hg2+) under ultraviolet radiation, good feasibility was

501

verified for Hg2+ detection. (C) Real-time monitoring of the fluorescence of EBCB,

502

with the successive addition of reagents. The inset shows the amplified range with an

503

enlarged x axis from 615 to 665 s, ultrafast response to Hg2+ was demonstrated. (D)

504

Optimization of probe sequence by the manner of signal-to-background ratio (F/F0).

505 506 507 508 24


Page 25 of 28


509

(A) Fluorescence

1.6e+6

500 nM

1.2e+6 [Hg 2+]

8.0e+5 4.0e+5

0

0.0 550 600 650 Wavelength¡¡/nm

(B) 15 F / F0

12

700

without Hg2+ add Hg2+

9 6 3 bla nk Li K Ag Mg Ca Sr Ba Mn Pb Zn Cu Cd Co N Sni Al Fe CH Cr 3 Hg

0 510 511

Figure 4. (A) Fluorescence spectra of EBCB responding to Hg2+ of different

512

concentrations. (B) Selectivity investigation by the manner of signal-to-background

513

ratio (F/ F0), where F0 was the fluorescence intensity of EBCB (100 nM) in the

514

presence of HP-4T (100 nM), and F was that with the addition of each ion. The

515

concentration of Hg2+ was 100 nM, and other ions were at 1 µM.

516 517 518 519 520 521 522 523 25



Page 26 of 28

524 525

Table 1．．Comparison of Our Proposed Method with Recently Reported Hg2+

526

Detection Methods.

527

strategy

detection time / min

detection limit /nM

Ref.

nitrogen-doped carbon quantum dots

NM&

230

31

HCR- and GO-based fluorescent sensor

140

0.3

19

5

10

32

20

7.2

33

20

0.15

34

40

0.024

35

NM&

125

23

60

11

24

0.5

0.82

this work

photoinduced electron transfer fluorescent probe BSA-stabilized Pt nanozyme for peroxidase mimetics cationic polymer/DNA interaction coupling hairpin DNA-scaffolded silver nanoclusters with ExoIII-assisted target recycling amplification thioether-based fluorescent covalent organic framework diketopyrrolopyrrole amphiphile-based micelle-like fluorescent nanoparticles target-lighted DNA-indicator 528

&

The detection time is not mentioned in the article.

529 530 531 532 533 534 535 536 537 538 539 540

26


Page 27 of 28


541 542

Table 2. Evaluation of Monitoring Mercury Pollution in Different real Water

543

Samples. sample river water

lake water

detected (nM)

ICP-MS

recovery

consistency

(meana ±SDb)

(nM)

(%)c

(%)d

0

5.47±0.0134

6.05

25

33.00±2.2429

35.35

110.10

93.34

50

53.01±4.5660

58.92

95.07

89.97

75

77.46±5.2903

81.37

95.98

95.20

100

102.99±7.7688

96.67

97.52

106.54

0

8.98±0.0446

8.08

25

30.87±0.1256

30.90

87.60

99.92

50

57.22±1.9666

62.50

96.49

91.56

75

82.05±3.0620

80.79

97.43

101.56

100

99.00±5.7475

102.06

90.03

97.01

spiked (nM)

90.54

111.06

544

a

Mean value of three-repeated detections. bStandard deviation. cRecovery percent

545

which was calculated by the formula (Cd-C0)/Cs, where C0 was the concentration of

546

the no-spiked sample measured by our approach, Cs was the spiked concentration, and

547

Cd was the concentration in each spiked sample measured by our approach.

548

d

549

the concentration measured by our approach, CICP-MS was that measured by ICP-MS.

Consistency percent which was calculated by the formula Cd/CICP-MS, where Cd was

550 551 552 553 554 555 556 557

27



558 559

(A) Black Glu Mg(NO3)2

Fluorescence

1.6e+6 1.2e+6

glucose GSH KI

8.0e+5 4.0e+5 0.0 500


(B)

560

550 600 650 Wavelength / nm

700

1.0 .8

Glu GSH KI Glu

.6 .4 GSH KI

.2 0.0 0

2 4 6 [antagonist] / µM

8

561

Figure 5. Screening mercury antagonists by the proposed strategy. (A) Fluorescence

562

spectra of EBCB and HP-4T after the incubation of Hg2+ (1.0 µM) with different

563

model molecules. (B) Demonstration of concentration-dependent antagonism. The

564

fluorescence intensity of the EBCB/HP-4T system in the presence of 1.0 µM Hg2+

565

was normalized as 1.0.

566 567 568 569 570 571 572

28


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A Target-Lighted dsDNA-Indicator for High-Performance Monitoring of

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