Colorimetric Aptasensor Based on Enzyme for the Detection of Vibrio

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Colorimetric aptasensor based on enzyme for the detection of Vibrio parahaemolyticus Shijia Wu, Yinqiu Wang, Nuo Duan, Haile Ma, and Zhouping Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03224 • Publication Date (Web): 24 Aug 2015 Downloaded from http://pubs.acs.org on August 25, 2015

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Journal of Agricultural and Food Chemistry

Colorimetric aptasensor based on enzyme for the detection of Vibrio parahaemolyticus

Shijia Wu, a Yinqiu Wang, a Nuo Duan,a * Haile Ma, b Zhouping Wang a a

State Key Laboratory of Food Science and Technology, Synergetic Innovation

Center of Food Safety and Nutrition, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China b

School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China

1

ACS Paragon Plus Environment


1

ABSTRACT

2

A simple colorimetric aptasensor system has been developed to detect Vibrio

3

parahaemolyticus. Magnetic nanoparticles (MNPs) are synthesized and conjugated

4

with specific aptamers against target and used as capture probes. In addition, this

5

method employs gold nanoparticles (AuNPs) as carriers of horseradish peroxidase

6

(HRP) and aptamers, which served as signal probes. In the presence of target, a

7

“sandwich-type” complex of AuNPs-HRP-aptamer-target-aptamer-MNPs is formed

8

through specific recognition of aptamers and corresponding target. As a result, HRP

9

molecules confine at the surface of the “sandwich” complexes catalyze the enzyme

10

substrate, 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2, and generate an optical

11

signal. Under optimal conditions, the signals are linearly dependent on V.

12

parahaemolyticus concentrations from 10 to 106 cfu/mL in a logarithmic plot, with a

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limit of detection 10 cfu/mL. Owning to AuNPs, a large amount of HRP could be

14

loaded; resulting in an amplified signal and the sensitivity would be improved. This

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strategy has the potential of being extended to the construction of simple monitor

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systems for a variety of biomolecules related to food safety.

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KEYWORDS: gold nanoparticles, magnetic nanoparticles, Vibrio parahaemolyticus,

18

horseradish peroxidase, colometric

19 20 21 22 23 24 25 2


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INTRODUCTION

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Vibrio parahaemolyticus, is a gram-negative bacterium, which naturally inhabits

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marine and estuarine environments. 1 It has become one of the most common causes

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of food-borne gastroenteritis, particularly in areas with high seafood consumption.

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Consumption of raw/undercooked seafood results in acute gastroenteritis with the

31

following symptoms: headache, abdominal cramps, vomiting, diarrhea and nausea.

32

With more and more consumption of seafood, V. parahaemolyticus becomes a food

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safety concern in many Asian countries.

34

cases of foodborne illnesses in China. In Chinese province of Guangdong, 29.22%

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outbreaks of food-borne disease were related to V. parahaemolyticus. 5 Therefore, it is

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very important to reduce contamination of seafood with V. parahaemolyticus in order

37

to prevent food poisoning and ensure the safety of seafood products.

4

2

3

V. parahaemolyticuscauses caused many

38

Recently, a variety of analytical methods have been reported for the detection of

39

pathogenic bacteria. Polymerase chain reaction (PCR), which is high sensitivity and

40

specificity, is a commonly used method for detection of V. parahaemolyticuscauses in

41

food and a real-time PCR have the quantitative function compared with the

42

conventional PCR. Besides, enzyme-linked immunosorbent assay (ELISA) is the

43

most popular and widely used method.

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test a large number of samples at the same time, and could be noticed by naked eyes,

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ELISA has become a powerful tool available for biological research and clinical

46

diagnostics. In addition, there are many other immunosensors, which was based on

47

antibody−antigen immunoreactions, such as electrochemical methods

48

fluorescence methods.

49

the quality of the antibodies used. The preparation of the antibodies via animal

50

immunization is time-consuming (several months), and the antibodies may become

10,11

6,7

Due to its convenient operation, ability to

8,9

and

However, these immunobioassays are heavily reliant on

3



51

susceptible to stability or modification issues.

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To rival antibodies in these ways, aptamers with high affinity and selectivity are

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beginning to emerge. Aptamers are short single-stranded DNA or RNA molecules

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which were selected through in-vitro selection or the systematic evolution of ligands

55

by exponential enrichment (SELEX).

56

advantages over antibody.

57

synthesis which is higher purity and lower costs. Aptamers can be flexibly modified

58

with various chemical tags which could not influence its affinity. Moreover, aptamers

59

are small in molecular weight and superior in stability, which can bear repetitious

60

denaturation and renaturation. Overall, these unique characteristics make aptamers an

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ideal recognition element for biosensors. As a potential analysis tool in the

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construction of aptasensors, optical analysis has attracted much more interest of

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researchers due to its high sensitivity, quick response and simple operation.

64

Nowadays, enzyme linked aptamer assay (ELAA) uses an aptamer as recognition

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element and enzyme as signal readout element has establishing different kinds of

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aptasensors.

67

bacteria application.

15,16

14

12,13

Aptamers possess many competitive

Aptamer can be routinely produced by chemical

However, there were seldom reports on ELAA in pathogenic

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Therefore, in this work, we designed an optical strategy for sensitive and specific

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quantitative detection of V. parahaemolyticus by gold nanoparticle-based enzyme-

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linked aptamer sandwich method. MNPs were modified with aptamer to act as the

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capture probe. AuNPs containing a large amout of HRP and aptamer were used as

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signal amplifiers. MNPs-aptamer-target-aptamer-HRP-AuNPs sandwich complexes

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would be formed based on the recognition of aptamers and target. In the addition of

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TMB-H2O2, HRP on the sandwich complexes were catalyzed and generated an optical

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signal. 4


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MATERIALS AND METHODS

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Materials

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Chloroauric acid (HAuCl4), streptavidin and 3,3,5,5-tetramethylbenzidine (TMB)-

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H2O2 were obtained from Sigma-Aldrich (U.S.A.). Horseradish peroxidase (HRP)

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was purchased from Sangon Biotechnology (Shanghai, China). Trisodium citrate, 1,6-

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hexanediamine, anhydrous sodium acetate, FeCl3·6H2O, glycol, 25% glutaraldehyde

82

(OHC(CH2)3CHO) and polyethylene glycol (PEG) were of analytical grade and were

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purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The V.

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parahaemolyticus aptamer were prepared in our laboratory

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purified by high-performance liquid chromatography (Sangon Biotechnology, Inc.,

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Shanghai, China). The sequence of V. parahaemolyticus aptamer was 5’-SH-

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TCTAAAAATGGGCAAAGAAACAGTGACTCGTTGAGATACT-3’ (apt 1), and

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5’-bio-TCTAAAAATGGGCAAAGAAACAGTGACTCGTTGAGATACT-3’ (apt 2).

17

and synthesized and

89 90

Instrumentation

91

Transmission electron microscopy (TEM) images were acquired with JEM–

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2100HR (TEM, JEOL Ltd., Tokyo, Japan). FT-IR spectra of nanoparticles were

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measured on a Nicolet Nexus 470 Fourier transform infrared spectrophotometer

94

(Thermo Electron Co., Boston, U.S.A.). UV-Vis spectra and absorbance were

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obtained using a UV-1800 spectrophotometer (Shimadzu Co., Kyoto, Japan).

96 97

Bacteria strains

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The V. parahaemolyticus ATCC 17802 was kindly donated by the Animal, Plant

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and Food Inspection Centre, Jiangsu Entry-Exit Inspection and Quarantine Bureau

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(Nanjing, China). V. parahaemolyticus was cultured in alkaline peptone with 3% 5



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NaCl (w/v) overnight past the logarithmic phase. One hundred microliters of the

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bacterial culture was diluted with medium and coated on the agar plates and cultured

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at 37 °C for 18 h to count colony forming units. The rest of the bacteria were

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collected carefully by centrifugation at 3000 rpm and 4 °C and washed twice in a

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1×binding buffer (50 mM Tris-HCl at pH 7.4, 5 mM KCl, 100 mM NaCl, and 1 mM

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MgCl2) at room temperature.

107 108

Synthesis of HRP-labeled AuNPs

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Briefly, all glassware used in the experiment were cleaned with aqua regia

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(HNO3/HCl, 3:1, v/v), rinsed thoroughly in ultrapure water, and dried prior to use.

111

0.5 mL of 1% HAuCl4 solution and 49.5 mL ultrapure water were heated to boiling

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point for 10 min with continuous stirring. Then, 1.5 mL of 1% trisodium citrate

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was rapidly added, stirred, and boiled for 15 min. The solution colour changed

114

from gray to blue, then purple, and finally to wine red during this period. The

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heating source was removed, and the colloid was cooled down to room temperature.

116

HRP-labeled AuNPs were prepared according to the previous report, with slight

117

modifications.

118

with 17.5 µL of HRP molecules (1 mg/mL) to incubate for 10 min at 25 °C under

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gentle shaking. Then, the mixed solution was stood overnight at 4 °C. After incubated

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with PEG (0.5%, w/v) for 30 min at 25 °C, the mixture was centrifuged (12 000 rpm,

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20 min) to remove the unbound HRP molecules and PEG, and washed with PBS for

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three times. The final deposition was suspended in 200 µL of PBS and stored at 4 °C

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for further use.

18

The prepared AuNPs (1 mL) was adjusted to pH 8.5 and mixed

124 125

Synthesis of aptamer functionalized HRP-labeled AuNPs 6


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HRP-labeled AuNPs modified by aptamer were prepared according to the literature 19

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with some modification.

This protocol was based on the Au-S interaction between

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the gold lattice and thiolated aptamer. Briefly, 10 µL of 10 µM apt 1 was added to 190

129

µL of the already prepared HRP-labeled AuNPs solution and reacted for 16 h. Then

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the HRP-labeled AuNPs-apt 1 complex was aged with salts (0.1 M NaCl, 10 mM

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phosphate, pH 7.0) for 40 h. The prepared complex was centrifuged at 12500 rpm for

132

15 min twice to remove the free aptamer. Then, the HRP-labeled AuNPs-apt 1 was

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dispersed in 200 µL of binding buffer for subsequent experiments.

134 135 136

Synthesis of MNPs and aptamer functionalized MNPs The amine-functionalized MNPs were prepared by a one-step solvothermal method 20

137

according to report.

Typically, 6.5 g of 1,6-hexanediamine, 2.0 g of anhydrous

138

sodium acetate and 1.0 g of FeCl3•6H2O were dissolved in 30 mL of glycol to form a

139

transparent solution with stirring vigorously at 50 °C. The mixture was subsequently

140

transferred into a Teflonlined autoclave and heated to 198 °C for 6 h. The product was

141

cooled to room temperature and then washed with water and ethanol (2 or 3 times)

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followed by drying at 50 °C.

143

The primary amine groups on the surface of MNPs were activated by

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glutaraldehyde, allowing amine groups on aptamer oligonucleotides to be covalently

145

attached. Briefly, 1 mg of the MNPs was dispersed in 1 mL of 10 mM phosphate

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buffer solution (PBS, pH 7.4), and 0.25 mL of 25% glutaraldehyde was added into the

147

solution. The reaction was continued for 2 h at room temperature gentle shaking,

148

followed by washing three times with PBS. The resultant MNPs were dispersed in 1

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mL of 10 mM PBS, and 50 µL of apt 2 (10 µM) were added and incubated for 2 h at

150

37 °C with gentle shaking. Next, the MNPs-apt 2 complexes were magnetically 7



151

collected and rinsed twice with PBS.

152 153

Procedure for analysis of V. parahaemolyticus

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In a typical experiment, 200 µL of AuNP-HRP-apt 1 and 150 µL of MNPs-apt 2 (1

155

mg/mL) were first mixed. Then, 50 µL of binding buffer containing various

156

concentrations of V. parahaemolyticus was added and incubated for 30 min at 37 °C

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with gentle shaking. After incubation, the magnetic beads were magnetically collected

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and rinsed with PBS twice. Then, 100 µL of TMB-H2O2 solution was added to each

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tube, and these tubes were incubated at 37 °C for 15 min with gentle shaking. Color

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development was stopped by adding 100 µL of 0.5 M sulfuric acid. After the

161

magnetic separation, the absorbance at 450 nm of the supernatant was read with UV

162

spectrophotometer.

163 164

Real samples treatment

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The water samples were collected from the Tai Lake, the Yellow sea, laboratory

166

and groundwater, China, respectively. Then the water samples were filtered through

167

0.45 µm filter. Different V. parahaemolyticus concentrations were then added to the

168

prepared samples for the experiments.

169 170

RESULTS AND DISCUSSION

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Principle of the aptasensor

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Scheme 1 shows the principle of this developed aptasensor. MNPs were modified

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with aptamer to act as the capture probe. AuNPs were coated with a large amout of

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HRP and linked to aptamer to act as signal amplifiers. In the presence of target,

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aptamers both on the surface of MNPs and AuNPs bound with the target with high 8


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affinity and specificity, leading to MNPs-aptamer-target-aptamer-HRP-AuNPs

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sandwich complexes formed. With an extra magnetic field, unbound AuNPs-HRP-

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aptamer was removed. In the addition of TMB-H2O2, HRP on the sandwich

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complexes were catalyzed and generated an optical signal. With the carrier of AuNPs,

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a large amount of HRP could be loaded; resulting in an amplified signal and the

181

sensitivity would be improved.

182 183

Characteristics of AuNPs and MNPs

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The morphology characteristics of AuNPs were shown in Fig. 1A. The AuNPs are

185

monodisperse and spherical with the average size of 15 nm. As shown in Fig. 1B, the

186

UV/vis spectrum of AuNPs solution (black line) exhibited a characteristic plasmon

187

absorption peak at 520 nm. After modification of HRP on the surface, the UV/vis

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spectrum of the AuNP complex showed a small surface plasmon shift from 520 nm to

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524 nm (red line). The shift after modification of AuNPs with HRP was attributed to

190

changes in the particle size and the dielectric nature surrounding the AuNPs due to the

191

presence of protein. Both results suggested successful immobilization of HRP onto

192

the AuNPs.

193

The TEM and FT-IR techniques were used to obtain information about the as-

194

synthesized MNPs. Fig. 1C shows TEM image of the MNPs, indicating a good

195

dispersibility and morphology. Fig. 1D shows the FT-IR spectra of MNPs. Formation

196

of MNPs were confirmed by a strong IR band at 586 cm-1 that comes from the Fe-O

197

vibrations. The bands around 1590, 1410 and 1060 cm-1 were NH bending mode and

198

C-N stretching vibration, respectively. The results from FT-IR revealed that the

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MNPs have been functionalized with amino groups in the synthetic process.

200 9



201

Characteristics of the nanoparticles conjugated to aptamer

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We applied UV/vis spectrophotometer to validate the successful coupling of

203

thiolated labeled aptamer (apt 1) to AuNPs-HRP through Au-S bonding and biotin

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labeled aptamer (apt 2) to avidin-modified MNPs through biotin-avidin specific

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bonding. As shown in Fig. 2A, AuNPs-HRP shows an absorbance peak at 524 nm

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(curve a), with the addition of apt 1 another peak at 260 nm which is characteristic

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absorption of DNA has been observed (curve b).

208

Similarly, no strong absorbance peak was detected for MNPs (curve c). After

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conjugation to avidin, a new absorption peak at approximately 280 nm, which is

210

characteristic absorption peak of avidin, was observed (curve d). With the subsequent

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addition of apt 2, the peak at 260 nm was also observed (curve e). These results

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demonstrate that aptamers were successfully coated on the nanoparticles.

213 214

Optimization for the assay

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According to the principle of the assay, experimental parameters such as the

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amount of aptamer functionalized MNPs and aptamer functionalized AuNPs-HRP

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complex would affect the signal response. The effect of the MNPs-apt 2 was first

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studied. As shown in Fig. 3A, Response signals increased with increasing MNPs-apt 2,

219

and a maximum was attained at 150 µL (1 mg/mL). Further increase in MNPs-apt 2

220

concentration, it had very little additional beneficial effect. Therefore, an optimal

221

volume of 150 µL MNPs-apt 2 was chosen in subsequent experiments. Fig. 3B shows

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optimization of the AuNPs-HRP-apt 1 complex concentration. The value of A450 was

223

found to increase as the AuNPs-HRP-apt 1 complex concentration was increased until

224

200 µL, after which the A450 plateaued and remained constant. Therefore, 200 µL of

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the AuNPs-HRP-apt 1 complex was used for subsequent experiments. 10


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Analytical performance

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Utilizing the optimal conditions in this system, the catalytic ability of the HRP-

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labeled AuNPs on TMB oxidation in the presence of different concentrations of V.

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parahaemolyticus was investigated. Results were evaluated in terms of (A-A0), where

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A0 and A are the absorbance of the colorimetric aptasenosr method in the absence and

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presence of V. parahaemolyticus, respectively. As displayed in Figure 4A, the

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absorbance was increased along with the V. parahaemolyticus concentration. A clear

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change from light yellow to dark yellow could be obviously differentiated by the

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naked eyes (inset A). As shown in Fig. 4B, the A-A0 exhibits a good linear

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relationship with V. parahaemolyticus in the concentration range from 10 to 106

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cfu/mL. The linear regression equation of V. parahaemolyticus is described as

237

Y=0.2357x-0.1057 (R2=0.9940), and the detection limit was found to be 10 cfu/mL.

238 239

Specificity, interference and practical performance of the assay

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To investigate the selectivity of the developed method, the AuNPs and MNPs

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complex were employed to detect other pathogenic bacteria, including V.

242

alginolyticus, V.vulnificus, V. mimicus, Escherichia coli, Salmonella typhimurium,

243

and Staphylococcus aureus at a concentration of 104 cfu/mL, which might affect the

244

detection of V. parahaemolyticus in real sample analysis. As shown in Fig. 5, it

245

clearly seen that only V. parahaemolyticus induces a dramatic increase of absorbance,

246

whereas other species produced signals as low as the blank control. These results

247

clearly demonstrated that the developed method is appropriate for the selective

248

detection of V. parahaemolyticus.

249

In addition, we examined the effect of a variety of possible interfering substances in

250

this system. We used a range of proteins, small molecules and ions to determine the 11



251

selectivity for V. parahaemolyticus. Under the optimized conditions (the

252

concentration of V. parahaemolyticus was set as 104 cfu/mL), none of the other

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interferents affected detection signals, even at concentrations as high as 2% bovine

254

serum albumin, 10 µg/mL of immunoglobulin G, and 100 mM of resorcinol,

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microcystic toxins and 500 mM of Na+, K+, Pb2+ and Cd2+. Thus results clearly

256

demonstrated that the developed method specifically identified V. parahaemolyticus

257

in real environment.

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The practical performance of the developed method was validated with four real

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water samples. Accuracy of this method was evaluated by determining the recoveries

260

of V. parahaemolyticus in the water samples by standard addition method. The water

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samples were spiked with V. parahaemolyticus at the desired concentrations (2×102,

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and 2×103 cfu/mL), which was analyzed by standard culture and colony counting

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method. Then, the spiked samples were analyzed by the colorimetric aptasensor

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method. As can be seen from Table 1, the recoveries of the V. parahaemolyticus in the

265

four real water samples are between 92.0% and 102.0%. These results show that

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recoveries of V. parahaemolyticus and the reproducibility are satisfactory.

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In summary, we developed a colometric aptasensor to detect V. parahaemolyticus

268

in which AuNPs were used as carriers of HRP and aptamer, and MNPs-aptamer were

269

used as supporting substrates for capture targets. The AuNPs-HRP-aptamer complex

270

loaded a high amount of HRP amplification enzyme, thus the developed assay based

271

on the AuNPs complex exhibited improved sensitivity. This feature, as well as its

272

convenient magnetic separation, made it a promising alternative to conventional

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pathogenic bacteria methods. This assay system displayed excellent specificity,

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sensitivity, and linearity for quantitative detection of the target molecules, along with

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the production of a color signal that can be detected by the naked eye. In addition, this 12


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method has a potential to detect other bacteria by changing aptamers.

277 278

AUTHOR INFORMATION

279

Corresponding Author

280

* Phone & Fax: +86-510-85917023; E-mail: [email protected]

281

282

Funding

283

This work was partialy supported by NSFC (31401576, 31401575), National

284

Science and Technology Support Program of China (2012BAK08B01), JUSRP11547,

285

and BK20140155.

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magnetite J.

nanoparticles

2006,

15


12,

and

hollow

6341-6347.


347

Figure captions:

348

Scheme 1. Schematic presentation of V. parahaemolyticus detection using

349

colorimetric aptasensor based on HRP

350

Fig.1 A TEM image of AuNPs (A), the UV/vis spectrum of the AuNPs and HRP

351

modified AuNPs (B), the TEM image (C), and the FT-IR spectrum of MNPs (D)

352

Fig. 2 Absorption spectra of bare AuNPs-HRP (a), apt 1-functionalized AuNPs-HRP

353

(b), bare MNPs (c), avidin conjugation to MNPs (d), and apt 2-functionalized MNPs

354

(e)

355

Fig. 3 Optimization of MNPs-apt 2 concentration (A), AuNPs-HRP-apt 1 complex

356

concentration (B). (concentration of V. parahaemolyticus was 104 cfu/mL)

357

Fig. 4 (A) Typical recorded output for the detection of different concentrations of

358

bacteria by the developed method. Inset is the color change by the naked eyes, (B)

359

Standard curve of the related absorbance (A-A0) versus the concentrations of bacteria.

360

Fig. 5 Specificity studies against other bacteria. Concentration of V. parahaemolyticus

361

was 104 cfu/mL, while the others was 105 cfu/mL.

362

Table 1 Analysis of V. parahaemolyticus cells in the spiked water samples by the

363

developed method.

16


Page 16 of 24

Page 17 of 24


Table 1 Analysis of V. parahaemolyticus cells in the spiked water samples by the developed method. Sample

Original

Spiked concentration

Measured concentration

Recovery

value(cfu/mL)

(cfu/mL)

(cfu/mL)

(%)

2

2

Tai Lake

0

2.0×10

(1.84±0.15)×10

92.0

seawater

0

2.0×103

(1.97±0.11)×103

98.5

laboratory

0

2.0×102

(1.91±0.28)×102

95.5

groundwater

0

2.0×103

(2.04±0.16)×103

102.0

17



Scheme 1.

18


Page 18 of 24


B

AuNPs HRP modified AuNPs

1.0

Absorbtion

Page 19 of 24

0.5

0.0

300

400

500

600

Wavelength (nm)

Fig.1

19


700


Fig. 2

20


Page 20 of 24

Page 21 of 24


Fig. 3

21



1.5

////

A

Page 22 of 24

concentration (cfu mL)

Absorption

6

10 5 10 4 10 3 10 2 10 10 blank

1.0

0.5

0.0 300

400

500

600

700

Wavelength (nm)

1.4

B

1.2 1.0

y=0.2357x-0.1057 2 R =0.9940

A-A0

0.8 0.6 0.4 0.2 0.0 1

2

3

4

5

6

Log Concentration of V. parahaemolyticus (cfu/mL)

Fig. 4

22


7

Page 23 of 24


Fig. 5

23



Graphic for table of contents

24


Page 24 of 24

Colorimetric Aptasensor Based on Enzyme for the Detection of Vibrio

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