Mimicking an Enzyme-Based Colorimetric Aptasensor for Antibiotic

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Article

Mimicking enzyme-based colorimetric aptasensor for antibiotic residue detection in milk combining magnetic Loop-DNA probes and CHA-assisted target recycling amplification Qian Luan, Ning Gan, Yuting Cao, and Tianhua Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02139 • Publication Date (Web): 27 Jun 2017 Downloaded from http://pubs.acs.org on June 28, 2017

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Journal of Agricultural and Food Chemistry

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Mimicking enzyme-based colorimetric aptasensor for antibiotic

2

residue detection in milk combining magnetic Loop-DNA probes

3

and CHA-assisted target recycling amplification

4

Qian Luana, Ning Gan*a, Yuting Caoa, Tianhua Lia

5

a

6

Materials Science and Chemical Engineering, Ningbo University, Ningbo, 315211, PR China

7

Email: [email protected]

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Tel: +86-574-87609987; Fax: +86-574-87609987

State Key Laboratory Base of Novel Functional Materials and Preparation Science, Faculty of

9 10

1

ACS Paragon Plus Environment


11

ABSTRACT

12

A mimicking enzyme-based colorimetric aptasensor was developed for the detection of kanamycin

13

(KANA) in milk using magnetic loop-DNA-NMOF-Pt (m-L-DNA) probes and catalytic hairpin

14

assembly (CHA)-assisted target recycling for signal amplification. The m-L-DNA probes were

15

constructed via hybridization of hairpin DNA H1 (containing aptamer sequence) immobilized

16

magnetic beads (m-H1) and signal DNA (sDNA, partial hybridization with H1) labeled nano

17

Fe-MIL-88NH2-Pt (NMOF-Pt-sDNA). In the presence of KANA and complementary hairpin

18

DNA H2, the m-L-DNA probes decomposed and formed an m-H1/KANA intermediate, which

19

triggered the CHA reaction to form a stable duplex strand (m-H1-H2) while releasing KANA

20

again for recycling. Consequently, numerous NMOF-Pt-sDNA as mimicking enzyme can

21

synergistically catalyze 3,3’,5,5’-tetramethylbenzidine (TMB) for color development. The

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aptasensor exhibited high selectivity and sensitivity for KANA in milk with a detection limit of

23

0.2 pg mL-1 within 30 min. The assay can be conveniently extended for on-site screening of other

24

antibiotics in foods by simply changing the base sequence of the probes.

25

Keywords: NMOF-Pt mimicking enzyme; loop DNA; antibiotics residue detection in milk;

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colorimetric aptasensor; CHA assisted signal amplification

27

2


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INTRODUCTION

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The negative influence of antibiotic residues in food has received widespread attention owing

30

to their abuse in agricultural practices.1 Therefore, there is an urgent need to develop new assays

31

with high sensitivity and selectivity for the detection of antibiotics in complex food matrices.

32

Towards that end, many assays have been developed for the determination of antibiotic residues

33

including electrochemical methods (EC),2 fluorescence methods (FL),3 high performance liquid

34

chromatography (HPLC),4 enzyme linked immunosorbent assay (ELISA),5 and colorimetric

35

methods.6,7 Among them, colorimetric methods have garnered considerable attention owing to

36

their unique advantages including fast screening, simple operation, and amenability towards visual

37

inspection.8 Generally speaking, most chromogenic approaches are based on the use of a

38

peroxidase enzyme to catalyze substrates such as 3,3’,5,5’-tetramethylbenzidine (TMB)9 and

39

2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonicacid) (ABTS)10 for color development. However,

40

natural enzymes suffer from intrinsic drawbacks such as low stability, susceptibility towards

41

inhibition, and time-consuming preparation.11 Notably, many researchers have found that some

42

materials with peroxidase-like properties can also catalyze substrates for color development, such

43

as ceria spheres (CeO2),12 platinum nanoparticles (Pt NPs),13 single-walled carbon nanotubes

44

(SWNTs),14 graphene,15 and metal organic frameworks (MOF),16,17 e.g., Fe-MIL-88NH2.18 Among

45

them, MOFs with high porosity, catalytically active sites, and specific surface areas have attracted

46

considerable interest and have been applied in the fabrication of mimicking enzyme probes in

47

colorimetric assays.19-21 Compared with the use of simple MOFs as probes, noble metal

48

nanoparticle functionalized-MOFs display superior catalytic and color-developing features owing

49

to synergistic effects. For example, Li’s group have successfully synthesized gold nanoparticles 3



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functionalized Fe-MOF through self-assembly, which exhibited significantly enhanced catalytic

51

activities

52

Au–SH–SiO2@Cu-MOFs hybrids as electrocatalysts for hydrazine determination.23 Inspired by

53

these examples, we synthesized mimicking-enzyme based composite probes based on platinum

54

nanoparticles (Pt NPs) functionalized nano Fe-MIL-88NH2 (NMOF-Pt) catalysts.

with

H2O2

than

that

of

Fe-MOF.22

Hosseini’s

group

constructed

55

Improving the specificity of colorimetric methods is important. Aptamers (Apt),

56

oligonucleotide sequences, have garnered considerable attention due to certain advantages such as

57

rapid synthesis in vitro, cost-effectiveness, and high affinities towards the target.24 Many

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aptamer-based biosensors have been successfully fabricated and most use DNA structures with

59

double helical conformations as probes (aptamers hybridize with their complementary DNA strand

60

sequences, abbreviated as cDNA) for antibiotic determination.25-27 The detection principle can be

61

summarized as a substitution reaction, which is driven by the higher affinity of the aptamer

62

towards the target than its complementary strand. However, some complementary double stranded

63

DNA (dsDNA) would unwound in difficulty leading to exhibit lower sensitivity due to higher

64

binding constants between the aptamer and cDNA than the target. Therefore, some secondary

65

structures comprised of double stranded nucleic acids with fewer base pairs between the aptamer

66

and cDNA have been designed to improve the binding efficiency between the aptamer and target.

67

Recently, secondary structures including hairpins,28 G-quadruplexes,29 and Y-DNA30 have been

68

applied in sensing platforms due to their unique structural features. Our group developed a highly

69

specific and sensitive Y-shaped DNA-based electrochemical aptasensor for antibiotic detection.31

70

Inspired by our success, in this study, a novel loop DNA (L-DNA) structure with structural

71

flexibility was designed through the partial complementarity of one molecular beacon to another 4


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ssDNA for antibiotic determination. The aptamer region was designed with a whole loop and

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sectional stem area of the molecular beacon to improve the binding efficiency between the target

74

and aptamer. Thus, the L-DNA structure may increase efficiency of the replacement reaction and

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have exceptional potential applications in food safety.32 In addition, in order to achieve higher sensitivity, some signal amplification technologies

76 have

78

amplification,34 and nicking enzyme-assisted target recycling.35 Our group developed colorimetric

79

aptasensor based exonuclease-I (Exo I)-assisted signal amplification and obtained satisfactory

80

specificity and sensitivity.36 However, such methods usually require expensive enzymes for

81

amplification, and unfortunately they are easily deactivated. Enzyme-free signal amplification

82

approaches are useful for target analysis, such as entropy-driven reactions,37 hybridization chain

83

reactions (HCR),38 and catalytic hairpin assembly (CHA).39 Among these methods, CHA has

84

served as a promising enzyme-free signal amplification strategy owing to the excellent

85

magnification, stability, and suitability.40 Its mechanism can be explained as follows: a pair of

86

designed hairpins can be triggered to form a duplex in the presence of a target, while the target is

87

released into the reaction system for signal amplification in the subsequent cycle. Chang’s group

88

also successfully developed a colorimetric sensing method based on the CHA reaction for protein

89

detection. In the presence of the target, H1 can hybridize with H2 to release the target for the next

90

reaction and the detection sensitivity can be improved to 2.3 pM.41 Therefore, the exploration of

91

the CHA strategy in colorimetric assays may result in potentially higher sensitivities and lower

92

costs for the detection of antibiotic residues in food.

93

been

established

including

polymerase

chain

reactions,33

77

exonuclease-assisted

In this work, a new mimicking enzyme based colorimetric sensor was designed for antibiotic 5



94

detection. NMOF-Pt nanotracer was used as the enzyme mimic and loop DNA (L-DNA)

95

functionalized magnetic beads were used to fabricate a composite probe (m-L-DNA). After the

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target replacement reaction, the magnetic capture DNA probes can trigger the hairpin assembly to

97

release the target for signal amplification. The fabrication process and detection mechanism were

98

as follows: first, the magnetic capture DNA probes (m-H1) were synthesized using capture DNA

99

(H1) containing an aptamer sequence fixed on the magnetic beads. The signal DNA probes

100

(NMOF-Pt-sDNA) were fabricated from signal DNA (sDNA) on the surface of NMOF-Pt. Finally,

101

m-L-DNA probes were formed by sDNA on the NMOF-Pt-sDNA by complete hybridization of

102

one part of the H1 on the m-H1. After the target was introduced, the m-H1/target intermediates

103

formed and the NMOF-Pt-sDNA hybrids were released into the supernatant. When H2, which was

104

completely complementary to H1, was added into the system, the m-H1/target intermediates

105

trigger CHA to form a more stable hybridized duplex between H1 and H2. Simultaneously, the

106

target is released into the reaction system for the next round, leading to a significant increase in

107

the amount of NMOF-Pt hybrids with highly peroxidase-like activity in the supernatant. Notably,

108

they can effectively catalyze the classical peroxidase substrate TMB in the presence of H2O2,

109

producing an obviously visible blue color. The developed mimicking enzyme-based colorimetric

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aptasensor eliminated the requirement for protein enzymes, making the system simple, stable, and

111

less expensive than conventional approaches; it exhibited high selectivity and sensitivity with a

112

detection limit of 0.2 pg mL-1. In addition, it has been successfully applied for the detection of

113

KANA in milk samples. The proposed mimicking enzyme based aptasensor presents a simple and

114

stable platform towards KANA detection and displays great potential in the detection of organic

115

residues in foods. 6


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MATERIALS AND METHODS

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Materials. Kanamycin (KANA), chloramphenicol (CAP), Streptomycin (STR), oxytetracycline

118

(OTC), chlortetracycline (CTC) and gentamicin sulfate (GS) were purchased from sigma (Milan,

119

Italy). Amino-functionalized magnetic beads (MB) and polyvinylpyrrolidone (PVP, Mw=55000),

120

2-aminoterephthalic acid (H2N-BDC) and phosphate buffer saline (PBS, pH 7.4, 0.1 M

121

KH2PO4-K2HPO4, 0.1 M KCl) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

122

Chloroplatinic

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3,3’,5,5’-tetramethylbenzidine (TMB) were obtained from Sinopharm Chemical Reagent Co., Ltd

124

(Shanghai, China). The blocking buffer solution 6-mercapto-1-hexanol (MCH), glutaraldehyde

125

(25% aqueous solution), tris (2-carboxyethyl) phosphine hydrochloride (TCEP), FeCl3 6H2O,

126

acetic acid, and sodium acetate were obtained from Aladdin Co., Ltd (Shanghai, China). All other

127

reagents were analytical grade and were used without further purification. And double-distilled

128

water was used throughout the study. All DNA oligonucleotides used in this paper were purchased

129

from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd (Shanghai, China)

130

and purified using high performance liquid chromatography, and the sequences of DNA

131

oligonucleotides were given below (the bold letters in H1 referred to the aptamer sequence of

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KANA)42:

133

H1: 5'-NH2-(CH2)6- CAA GAT GGG GGT TGA GGC TAA GCC GAA CCC TTT TGT TTT TT

134

-3'

135

H2: 5'-AAA AAA CAA AAG GGT TCG GCT TAG CCT CAA CCC CCA TCT TG -3'

136

sDNA: 5'-SH-(CH2)6- TTT AAA CAA AAC CAT CTT G-3'

acid

(H2PtCl6,

37

wt%

Pt),

hydrogen

peroxide

137 7


(H2O2,

30%)

and


138

Instruments. The transmission electron microscopic (TEM) images were obtained with a H600

139

transmission electron microscope (Hitachi, Tokyo, Japan). Infrared spectra were recorded with a

140

Nicolet 6700 FT-IR spectrophotometer (Madison, Wisconsin, USA). The UV-Vis spectrums were

141

recorded on a UV-1800 spectrophotometer (Shimadzu, Japan). The powder X-ray diffraction

142

(PXRD) characterization was performed using X-ray diffraction (Bruker, D8 Focus, Karlsruhe,

143

Germany) with Cu Kα radiation at room temperature. DNA analysis was performed by microchip

144

electrophoresis system (MCE-202 MultiNA, Shimazu, Japan). Dynamic Light Scattering (DLS)

145

images were carried out by the Malvern zetasizer Nano ZS90, Malvern instruments Ltd., (Malvin

146

Co., UK).

147 148

Synthesis of NMOF-Pt. NMOF-Pt was prepared with partial modification according to a method

149

reported in the literature43. Briefly, 12 mL as-synthesized Pt NPs (synthesized details in supporting

150

information) solution (0.6 mM) was dropwise added into 12 mL DMF of Fe-MIL-88NH2

151

(synthesized details in supporting information) under vigorous stirring (750 rpm/min), and then

152

the mixed solution was further stirred at room temperature for 2 h. Subsequently, NMOF-Pt was

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collected by centrifugation at 8000 rpm for 10 min and washed twice with 10 ml ethanol and DMF.

154

Finally, the obtained NMOF-Pt was dried in a vacuum oven at 60 ºC overnight.

155

Synthesis of capture DNA probes. The capture DNA probes consisting of H1 and

156

amino-functionalized magnetic beads (MB) were prepared according to the procedure of literature

157

with further modification44. 2 mg MB was suspended in 2 mL PBS buffer solution containing

158

2.5% glutaraldehyde and sonicated to obtain a homogeneous solution, then shook 1 h at room

159

temperature. Furthermore, 50 µL 10 µM H1 was firstly denatured by heating for 5 min at 95 °C in 8


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a water bath and cooled to room temperature, then H1 was added into the mix-solution stirring

161

another 8 hours. After completion of the reaction, the excess H1 was removed by magnetic

162

separation. And the precipitation was washed for three times using 10 mL PBS buffer solution.

163

Finally, the obtained m-H1 was dissolved in 1 mL PBS containing 1mM MCH to block the

164

nonspecific binding sites on the m-H1 and the capture DNA probes were saved at 4°C for further

165

use.

166

Synthesis of signal DNA probes. The prepared progress of signal DNA probes (NMOF-Pt-sDNA)

167

referred to the literature45. 10 µL of 10 µM signal DNA (sDNA) was incubated with 1 µL of

168

10mM TCEP for 1h to reduce disulfide bonds at room temperature. And then 100 µL 0.5 mg mL-1

169

NMOF-Pt was introduced into the solution for forming the NMOF-Pt-sDNA at 4°C overnight.

170

Next, the solution was centrifuged to remove the un-bonded free sDNA at 8000 rpm 10 min and

171

the acquired precipitate was washed three times with 1 mL PBS buffer. Finally, the

172

NMOF-Pt-sDNA was re-dispersed 100 µL PBS buffer containing 1mM MCH to block the

173

nonspecific binding sites on the NMOF-Pt-sDNA and was stored at 4°C for further use.

174

Synthesis of m-L-DNA probes. 100 µL capture DNA probes (m-H1) was incubated with 100 µL

175

signal DNA probes (NMOF-Pt-sDNA) 2h at 37°C to form the m-L-DNA probes

176

(m-H1-sDNA-Pt-NMOF). Then, the precipitate (m-H1-sDNA-Pt-NMOF) was collected through

177

magnetic separation and was washed three times by PBS buffer to remove the unbounded

178

NMOF-Pt-sDNA. Lastly, the m-L-DNA probes were dissolved 200 µL PBS for further use.

179

Procedure for Analysis of KANA. For the determination of KANA, 50 µL m-L-DNA probes

180

were incubated with 50 µL KANA with different concentrations and 10 µL 10 µM H2 at ambient

181

for 30 min. After magnetic separation, 50 µL supernatant was extracted to react with 1mM TMB, 9



182

25mM H2O2 and 40 mM NaAc buffer solution (pH 4.0) at 45 °C for 10 min in dark . Finally, the

183

absorbance of the mixture was determined at 650 nm by UV-1800 spectrophotometer for

184

quantitative analysis.

185

Real Sample Treatment. In order to verify the applicability in real sample, three milk samples

186

with different brands of Jindian, Telunsu and Wangzai from Jiabei supermarket (Ningbo, China)

187

were assayed through the developed colorimetric strategy. The milk samples were pre-treated

188

according to the ELISA method to remove the adipose by centrifuging with 10000rpm 10 min at

189

10°C. And the treated milk samples were diluted about 500 times of PBS buffer. Then, different

190

KANA concentrations (0.005ng mL-1, 0.50 ng mL-1, 5.0 ng mL-1) were added into the milk

191

samples for experiments. The final milk samples were measured in terms of the progresses

192

mentioned above.

193 194

RESULTS AND DISCUSSION

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Characteristics of Capture DNA Probes and Signal DNA Probes. Magnetic beads (MB) and

196

capture DNA probes (m-H1) were characterized by energy dispersive X-ray spectroscopy (EDX),

197

FT-IR spectroscopy, dynamic light scattering (DLS), and ultraviolet–visible (UV–vis)

198

spectrophotometry. FT-IR was utilized to verify the existence of amino-groups (-NH2) on the

199

commercial MB (600 nm; the DSL image is shown in Figure S1A in the supporting information);

200

the result is shown in Figure S1B. As shown in Figure S1B, the strong band at around 575 cm−1

201

corresponded to the vibrations of Fe-O.46 The characteristic absorption bands at 1599 cm−1 and

202

3463 cm−1 were ascribed to the bending and stretching vibrations of the N-H bond, respectively.47

203

M-H1 was characterized by EDX and UV-Vis. As is shown in Figure 1A, characteristic peaks of 10


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204

m-H1 appeared in the EDX image, including those corresponding to C, N, O, Fe, and P. The peak

205

of P may be attributed to H1, indicating the successful immobilization of H1 on MB. The UV-vis

206

absorption spectra of MB, H1, and m-H1 are shown in Figure 1B; bare MB exhibited no obvious

207

absorption from 220 nm to 750 nm (curve a), and bare H1 exhibited a characteristic peak at 260

208

nm (curve b). Compared with curve a, a new absorption peak appeared at 263 nm in curve c,

209

which was ascribed to the DNA H1. The results indicated that H1 was successfully fixed on the

210

MB, forming the capture DNA probes (m-H1).

211 212

Preferred position for Figure 1

213 214

Meanwhile, the formation of signal DNA probes (NMOF-Pt-sDNA) was confirmed by TEM,

215

XRD, EDX, and UV-vis. TEM images of NMOF, NMOF-Pt, and Pt NPs are shown in Figure 2A,

216

2B, and S2A, respectively. Bare NMOF showed an unusual octahedron morphology with an

217

average diameter of approximately 378 nm (Figure 2A; the DLS image is shown in Figure S2B).

218

When the 2.5 nm Pt NPs (the DLS image is shown in Figure S2C) were coated on the NMOF,

219

numerous star-like dots were uniformly dispersed on the surface of NMOF with size from 378 nm

220

increased to 386 nm (Figure S2D). Moreover, to verify the stability of NMOF-Pt, photos of 100

221

µL 0.5 mg mL-1 NMOF-Pt aqueous solution at ambient temperature before (E) and after (F) 30

222

min were obtained (Figure S2E and S2F); there was no precipitate and the particles remained

223

homogeneous after 30 min. Figure 2C shows the high magnified TEM image of NMOF-Pt, where

224

dots can be more clearly observed on the surface of NMOF. Moreover, powder X-ray diffraction

225

(PXRD) (Figure S3) indicated that the particles were highly crystalline and the XRD patterns of 11



226

the as-synthesized NMOF matched well with the reported XRD patterns of Fe-MIL-88NH2.48 In

227

order to confirm that the Pt NPs and sDNA were successfully labeled on the surface of NMOF,

228

energy dispersive X-ray spectroscopy (EDX) was performed (Figure 2D; the inset shows the

229

corresponding SEM image). Peaks corresponding to C, N, O, P, Pt, and Fe were observed. The

230

dots on the NMOF might explain the presence of Pt, as they might be Pt NPs. Furthermore, the

231

peak corresponding to phosphorus might be attributed to the immobilization of sDNA. The

232

fabrication of NMOF-Pt-sDNA was also confirmed by Fourier transform infrared spectroscopy

233

(Figure S4). Bare NMOF exhibited similar characteristic bands to reference documents (curve

234

a).49 Comparing curve b with curve a, the new peak at 1041 cm-1 may be attributed to the

235

phosphate groups and ribose.50,51 Overall, the above results demonstrated the successful formation

236

of signal DNA probes (NMOF-Pt-sDNA).

237 238


239 240

Verification of the Configuration of L-DNA. To obtain a stabile L-DNA structure, three

241

different nucleotide lengths (15 nt, 17 nt, and 19 nt) of sDNA were designed to complement H1

242

and the products were assayed by electrophoresis. As is shown in Figure S5, bands corresponding

243

to pure H1, 15 nt sDNA, 17 nt sDNA, and 19 nt sDNA appeared in lanes 1, 2, 3, and 4,

244

respectively. After mixing H1 with the three different lengths of sDNA, the electrophoresis stripes

245

appeared in lanes 5, 6, and 7. There were two bands corresponding to H1 and sDNA with 15 nt in

246

lane 5, and the two bands in lane 6 also corresponded to the H1 and sDNA with 17 nt, possibly

247

indicating that H1 did not combine with sDNA with 15 nt and 17 nt. When sDNA with 19 nt was 12


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248

mixed with H1, new and darker electrophoresis stripe appeared in lane 7, which revealed the

249

formation of L-DNA (hybrid of H1-sDNA). Therefore, we chose sDNA with 19 nt for subsequent

250

experiments.

251

In order to verify the L-DNA configuration and the feasibility of the CHA amplification

252

reaction, seven experiments were carried out and the DNA products were analyzed by microchip

253

gel electrophoresis (Figure 3). Bands corresponding to bare H1, sDNA, and H2 appeared in lanes

254

1, 2, and 3, respectively. After H1 was incubated with sDNA, a delayed band (33 bp) appeared in

255

lane 4, indicating the successful formation of L-DNA. In the presence of 30 ng mL-1 KANA, the

256

band corresponding to 33 bp was lighter than that in lane 4, which indicated some of the L-DNA

257

was unwound due to the higher affinity between H1 and KANA than sDNA (lane 5). After adding

258

a high concentration of KANA (30 µg mL-1), the band at 33 bp disappeared, which may be

259

attributed to the destruction of L-DNA (lane 6). When KANA and H2 were simultaneously

260

introduced into the system, a higher and darker band (45 bp) corresponding to the H1-H2 hybrid

261

product was observed in lane 7, implying that the CHA was successful triggered in the presence of

262

the target and H2. These results confirmed the successful configuration of the L-DNA and CHA

263

assisted target recycling, which proved the feasibility of the signal amplification for the

264

colorimetric aptasensor.

265 266


267 268

Amplification Performance of NMOF-Pt and m-L-DNA Probes. To verify the signal

269

amplification performance of the NMOF-Pt and m-L-DNA probe-based CHA-assisted target 13



270

recycling, a series of experiments were designed for KANA determination (Figure 4). In the

271

absence of the target, NMOF-Pt-sDNA and NMOF-sDNA reacted with m-H1 (curves a and b,

272

respectively). There was no absorbance at 650 nm (TMB’s chromogenic peak with H2O2) due to

273

the lack of the signal DNA probes in the supernatant. Curves c and d demonstrated that 30 ng mL-1

274

KANA was incubated with m-H1-sDNA-NMOF and m-H1-sDNA-Pt NPs, which displayed

275

obvious signals at 650 nm. After applying the NMOF-Pt-labeled signal DNA probes to synthesize

276

the m-L-DNA probes, there was a significant increase (3.2 fold) in the absorbance at 650 nm of

277

curve e compared to that of curve c, indicating the good signal amplification performance of Pt

278

NPs; the results were consistent with that of the previous work.13 When H2 was introduced into

279

the system, a much stronger signal at 650 nm appeared because the CHA reaction was triggered to

280

facilitate H1-H2 hybridization while releasing the target for next recycling (curve f). A 12-fold

281

higher signal intensity was observed as compared to curve c. These results implied that NMOF-Pt

282

functionalized m-L-DNA probes have enzyme-mimicking features and the aptasensor based CHA

283

reaction can significantly amplify the signals.

284 285


286 287

Optimization of the Aptasensor. To obtain remarkable signals for KANA detection, the reaction

288

conditions, including the concentrations of NMOF-Pt, ratio of capture DNA probes to signal DNA

289

probe, incubation time, temperature, and pH of the m-L-DNA probes with the targets, were

290

optimized. The concentration of the NMOF-Pt nanotracer affected the signal of the aptasensor;

291

therefore, different concentrations of NMOF-Pt were used to label the sDNA. Upon adding 14


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292

different concentrations (0.2 to 0.8 mg mL-1) of NMOF-Pt, the absorbance increased with

293

increasing concentrations of NMOF-Pt until 0.5 mg mL-1 NMOF-Pt, where the absorbance

294

plateaued. Therefore, 0.5 mg mL-1 NMOF-Pt was selected for KANA detection. Different ratios of

295

the capture DNA probes (m-H1) to nanotracer (NMOF-Pt-sDNA) (0.25, 0.5, 1.0, 1.5, 2.0) were

296

also evaluated (Figure S6B). There was little change in the signal intensity when the ratio was

297

greater than 1.0, therefore the ratio of 1.0 was chosen for subsequent experiments. The optimal

298

incubation temperature (15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C), time (10 min, 20 min, 30 min,

299

40 min, 50 min), and pH (5.0, 6.0, 7.0, 7.4, 8.0, 9.0) were also determined (Figure S7).

300

Accordingly, 25 °C, 30 min, and pH 7.4 were selected for target detection. In addition, the

301

concentrations of TMB and H2O2 were optimized (Figure S8). The maximum absorbance (650 nm)

302

was obtained in the presence of 1 mM TMB in the range from 0.6 to 1.6 mM (Figure S8A) and 25

303

mM H2O2 in the range of between 15 and 35 mM (Figure S8B), respectively. Therefore, 1 mM

304

TMB and 25 mM H2O2 were determined to be optimal for the signal readout.

305

Analytical Performance. To investigate the analytical performance of the developed aptasensor,

306

different concentrations of KANA were analyzed under the optimized reaction conditions. As

307

shown in Figure 5A, the absorbance at 650 nm increased with increasing concentrations of KANA

308

from 0.0005 to 30 ng mL-1. The corresponding color change could also be observed by the naked

309

eye (inset in Figure 5A), as the blue color deepened with increasing concentrations of KANA.

310

Figure 5B shows the calibration plots of different concentrations of KANA and the corresponding

311

absorbance at 650 nm; the linear curve fit a regression equation of y = 0.6916 lg CKANA+0.1787

312

(R2 = 0.993, n = 3). The proposed mimicking enzyme-based colorimetric aptasensor displayed

313

high sensitivity with a detection limit of 0.2 pg mL-1 (S/N = 3). 15



314 Preferred position for Figure 5

315 316 317

In addition, the detection range and sensitivity of some recently reported methods for KANA

318

detection are listed in Table 1 for comparison. Among the available detection methods, our probe

319

exhibited a lower detection limit, and required a shorter reaction time of 30 min. Moreover, the

320

detection range and sensitivity of some colorimetric methods based on silver nanoparticles or

321

using gold nanoparticles as mimicking enzyme for KANA detection were also compared. Our

322

probe exhibited a lower detection limit. The lower detection limit and shorter reaction time were

323

ascribed to the mimicking enzyme NMOF-Pt-labeled m-L-DNA probes based on the CHA

324

amplification strategy.

325 326

Preferred position for Table 1

327 328

Specificity, Reproducibility, Stability, and Practical Performance of the Aptasensor. To

329

determine the specificity and selectivity of the developed aptasensor, 30 ng mL-1 KANA, five

330

other antibiotics (60 ng mL-1 CAP, STR, OTC, CTC, and GS), 100 ng mL-1 metal ions (Zn2+, Ca2+,

331

and Mg2+), amino acids (valine, methionine, isoleucine), and proteins (casein, globulin, albumin)

332

which may coexist in milk were mixed and assessed. From Figure 6A, the potential interfering

333

substances had nearly no absorbance at 650 nm and observed colorless solution by the naked eye;

334

the signal intensity of KANA was basically the same as that of the mixture which appeared bright

335

blue by the naked eye (inset). This may be attributed to the high affinity between the target and 16


Page 16 of 36

Page 17 of 36


336

aptamers. These results demonstrated the high specificity and selectivity of the developed

337

aptasensor for KANA detection.

338

Moreover, the reproducibility of the aptasensor was evaluated from the relative standard

339

deviation (RSD), which was measured six times using 30.0 ng mL-1 KANA; the obtained RSD

340

was about 2.6%. The satisfactory RSD indicated that the proposed strategy has good

341

reproducibility. The intra-day and inter-day precisions and accuracies were assessed using three

342

KANA samples (0.01 ng mL-1, 1.00 ng mL-1, 10.0 ng mL-1) and five replicates; the results are

343

shown in Table S1. The inter-day and intra-day precision was less than 6.85%, and the accuracies

344

ranged from 96.3% to 112%, indicating acceptable reproducibility, precision, and accuracy.

345

The stability of the proposed biosensor was evaluated after the m-L-DNA probes were stored

346

at 4 °C for 5 to 30 days (Figure 6B). The absorbance was determined after incubating 30 ng mL-1

347

KANA with m-L-DNA probes for different amounts of time. The absorbance hardly decreased

348

after storing for 30 days (curve b) as compared to the freshly prepared m-L-DNA probes (curve c),

349

and the signal intensity was nearly 91% of its initial value (the initial absorbance was 1.04 and the

350

absorbance after 30 days was 0.95), indicating suitable stability.

351 352


353 354

In order to verify the practical performance of the aptasensor, three milk samples (Jindian

355

(sample 1), Telunsu (sample 2), and Wangzai (sample 3)) from Jiabei supermarket (Ningbo, China)

356

were assayed for KANA and the results were compared with those of the commercial ELISA

357

method (Table 2). The results of the blank milk samples were obtained using the proposed method. 17



358

The results from the developed aptasensor were almost identical to those of the commercial

359

ELISA method (Table 2). The recovery of KANA was also estimated via the standard addition

360

method, in which a known quantity of KANA (0.005, 0.500, and 5.00 ng mL-1, respectively) was

361

introduced. The recoveries from three milk samples were between 90.0% and 124%, and the

362

relative standard deviation (RSD) was acceptable. The results clearly demonstrated that the

363

developed aptasensor can be applied for KANA detection in complex samples.

364 Preferred position for Table 2

365 366 367

In summary, mimicking enzyme NMOF-Pt-based colorimetric aptasensor using a

368

combination of magnetic L-DNA probes and CHA-assisted target recycling was successfully

369

designed and applied for KANA detection in milk. The NMOF-Pt hybrids on the m-L-DNA

370

probes with high peroxidase-like activity can effectively catalyze the H2O2-TMB system for color

371

development owing to the synergetic effect between the NMOF and Pt NPs. Furthermore, in the

372

presence of the target and H2, the CHA reaction can form stable m-H1-H2 hybrids with

373

concomitant release of the target into the supernatant for recycling, leading to numerous signal tag

374

NMOF-Pt in the supernatant for signal amplification. Notably, the developed procedure does not

375

require any enzymes, and can be carried out at room temperature. Moreover, the developed

376

biosensor was applied in real milk samples. The aptasensor could be expanded to detect other

377

antibiotics by changing the corresponding aptamer sequence on the capture DNA probes.

378

Therefore, the assay offers broad prospects for early diagnosis and assessment of antibiotic

379

residues in food.

380

18


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381

ABBREVIATIONS USED

382

CHA= catalytic hairpin assembly

383

L-DNA=loop DNA

384

KANA= kanamycin

385

NMOF-Pt =nano Fe-MIL-88NH2-Pt

386

sDNA=signal DNA

387

m-H1= H1 labeled magnetic beads

388

nt = nucleotide

389

bp= base pairs

390

TMB=3,3’,5,5’-tetramethylbenzidine

391

ABTS=2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonicacid)

392

EC= electrochemical method

393

FL= fluorescence method

394

HPLC =high performance liquid chromatography

395

ELISA= enzyme linked immunosorbent assay

396

Apt=aptamer

397

HCR= hybridization chain reaction

398

CAP= chloramphenicol

399

STR= streptomycin

400

OTC= oxytetracycline

401

CTC= chlortetracycline

402

GS= gentamicin sulfate 19



403

MB= magnetic beads

404

PVP= polyvinylpyrrolidone

405

MCH=6-mercapto-1-hexanol

406

TCEP= tris (2-carboxyethyl) phosphine hydrochloride

407

SEM= Scanning electron micrographs

408

TEM= transmission electron microscopic

409

FT-IR= Fourier transform infrared

410

PXRD= powder X-ray diffraction

411

DLS= Dynamic Light Scattering

412

PBS= phosphate buffer saline

413

UV–vis= Ultraviolet–visible

414

RSD= relative standard deviation

415

20


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416

ASSOCIATED CONTENT

417

Supporting Information

418

Fig. S1. DLS image (A) and FT-IR spectrum (B) of MB. Fig. S2. (A) TEM image of Pt NPs; DLS

419

of NMOF (B), Pt NPs (C) and NMOF-Pt (D); the photos of 100 µL 0.5 mg mL-1 NMOF-Pt

420

aqueous solution before (E) and after (F) standing for 30 min. Fig. S3 The XRD spectrum of

421

NMOF. Fig. S4. FT-IR spectrum of NMOF (a) and NMOF-Pt-sDNA (b). Fig. S5. The simulated

422

gel electrophoresis results of H1 (lane 1), 15 nt sDNA (lane 2), 17 nt sDNA (lane 3), 19nt sDNA

423

(lane 4), reaction of H1 and 15 nt sDNA (lane 5), reaction of H1 and 17 nt sDNA (lane 6) and

424

reaction of H1 and 19 nt sDNA (lane 7). Lane 8 shows the 25 bp ladder. Fig.S6. The effects of the

425

concentration of NMOF-Pt (A) and the ratio of capture DNA probes to signal DNA probes (B).

426

Fig.S7. The effects of reaction temperature (A), time (B) and pH (C). Fig.S8. Effects of the

427

concentrations of TMB (A) and H2O2 (B). Table S1 The precision and accuracy of Intra-day and

428

Intrer-day for KANA detection.

429 430 431

AUTHOR INFORMATION

432

Corresponding Author

433

*Phone: 86-574-87609987, E-mail: [email protected]

434 435

Funding

436

This work was supported by the National Natural Science Foundation of China (No. 31070866),

437

the Natural Science Foundation of Zhejiang (LY17C200007 and LY16B050003), the Natural 21



438

Science Foundation of Ningbo (2016A610084), and the K.C. Wong Magna Fund in Ningbo

439

University.

440

Notes.

441

The authors declare no competing financial interest.

22


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442

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443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484

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610 611

26


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1


FIGURE CAPTIONS

2 3 4

Scheme. Scheme of depicting the developed colorimetric sensor for kanamycin detection based on the NMOF-Pt labeled magnetic L-DNA probes and CHA amplification.

5 6

1



7

Fig. 1

8 9 10

Figure 1 (A) EDX image of capture DNA probes (m-H1); (B) The UV-vis spectra of magnetic beads (MB) (a), H1 (b) and capture DNA probes (m-H1) (c).

11 12

2


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13

Fig. 2

14 15 16

Figure 2 TEM images of NMOF (A), NMOF-Pt (B) and NMOF-Pt with high high magnification (C); (D) EDX spectrum of NMOF-Pt-sDNA.

17 18 19 20 21 22

3



23

Fig. 3

24 25 26 27 28 29

Figure 3 The simulated gel electrophoresis analysis of the formation of L-DNA and the verification of CHA amplification. Lane 1: 1.0 μM H1 (41 nt); Lane 2: 5.0 μM sDNA (19 nt); Lane 3: 1.0 μM H2 (41 nt); Lane 4: reaction containing H1 and sDNA; Lane 5: reaction of H1 and sDNA with KANA (30 ng mL-1); Lane 6: reaction of H1 and sDNA with KANA (30 μg mL-1); Lane 7: reaction H1 and sDNA with KNAN (30 ng mL-1) and H2; Lane 8: 25-bp ladder.

30 31

4


Page 30 of 36

Page 31 of 36

32


Fig. 4

33 34 35 36 37 38

Figure 4 The verification of NMOF-Pt and CHA for signal amplification. (a) blank (m-L-DNA probes without KANA); (b) reaction of NMOF labeled L-DNA without KANA; (c) reaction of NMOF labeled L-DNA with KANA; (d) reaction of Pt NPs labeled L-DNA with KANA; (e) reaction of NMOF-Pt labeled L-DNA with KANA; (f) reaction of NMOF-Pt labeled L-DNA with KANA and H2.

39

5



40

Fig. 5

41 42 43 44 45

Figure 5 (A) UV-vis absorption spectra of the proposed aptasensor in the presence of different concentrations of KANA target (the inset images shows the corresponding digital camera pictures of colorimetric responses); (B) The relationship between KANA concentration and UV absorbance intensity at 650nm with the developed colorimetric sensor.

46 47 48 49 50 51 52 53

6


Page 32 of 36

Page 33 of 36


54

Fig. 6

55 56 57 58 59 60

Figure 6 (A) Specificity and selectivity against other antibiotics, metal ions, amino acids and proteins which might coexist in milk samples (the inset images shows corresponding digital camera pictures); (B) The stability of the developed colorimetric aptasensor. The UV-vis spectrum of blank (a), reaction of m-L-DNA after storing 30 days and KANA (b), reaction of fresh prepared m-L-DNA and KANA (c).

61

7



62 63

Page 34 of 36

Table 1. Table 1 The assay compared to the other methods for KANA detection. The concentration of KANA, (ng mL-1)

Reaction Time

Method

Impedimetric sensor

Reference Linear range

Detection Limit

(min)

1.2-75

0.11

60

52

Voltammetric aptamer sensor Chemiluminescent aptasensor Electrochemical aptasensor

0.01-150

0.005

60

53

0.005-0.5

0.002

45

54

0.005-40

0.0046

50

55

colorimetric method

50-600

2.6

20

56

colorimetric biosensor

1-100 nM

1.49 nM

30

57

colorimetric biosensor

0.1-20 nM

0.1 nM

45

58

Colorimetric aptasensor

0.0005-30

0.0002

30

This work

64 65 66

8


Page 35 of 36

67


Table 2. The developed method and ELISA method for KANA detection ( x ±s, n=5). Samples number

Sample 1

Sample 2

Sample 3

68 69 70

Blank (ng mL-1)

Added (ng mL-1)

Proposed method (ng mL-1)

ELISA (ng mL-1)

Recovery (%)

RSD (%)

0

0.005

0.0045±0.0010

-

90.0

2.75

0

0.500

0.508±0.019

0.50±0.02

101.6

1.28

0

5.00

4.92±0.025

5.05±0.031

98.4

2.09

0.116

0.005

0.114±0.023

0.11±0.02

94.2

1.65

0.116

0.500

0.623±0.031

0.62±0.038

101.1

1.58

0.116

5.00

5.35±0.012

5.02±0.015

104.6

1.26

0

0.005

0.0062±0.0015

-

124

3.15

0

0.500

0.511±0.020

0.50±0.035

102.2

2.03

0

5.00

5.18±0.018

5.09±0.017

103.6

2.27

Note: The symbol ‘- ’means no detected by the corresponding method.

9



71 72 73

TOC Scheme of depicting the developed colorimetric sensor for kanamycin detection based on the NMOF-Pt labeled magnetic L-DNA probes and CHA amplification.

74 75

10


Page 36 of 36

Mimicking an Enzyme-Based Colorimetric Aptasensor for Antibiotic

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