An electrochemical aptasensor electrocatalyst for detection of thrombin

Accepted Manuscript An electrochemical aptasensor electrocatalysts for detection of thrombin Rong Tian, Xiaojun Chen, Qingwen Li, Cheng Yao PII:

S0003-2697(16)00044-0

DOI:

10.1016/j.ab.2016.01.021

Reference:

YABIO 12299

To appear in:

Analytical Biochemistry

Received Date: 7 November 2015 Revised Date:

25 January 2016

Accepted Date: 28 January 2016

Please cite this article as: R. Tian, X. Chen, Q. Li, C. Yao, An electrochemical aptasensor electrocatalysts for detection of thrombin, Analytical Biochemistry (2016), doi: 10.1016/j.ab.2016.01.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

An electrochemical aptasensor electrocatalysts for detection of

2

thrombin

3

Rong Tiana,b*, Xiaojun Chena, Qingwen Li b, Cheng Yaoa,*

4

a

5

China

6

b

RI PT

Suzhou institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou, 215123, P. R. China

8

10

Subject category: Enzymatic Assays and Analyses Short title: aptasensor based on nanomaterial

11

M AN U

9

12

*Corresponding authors

13

Tel. &Fax: +86-25-58139482

14

E-mail address: [email protected] (R Tian),

18 19 20 21 22 23 24 25 26 27 28 29

TE D

17

EP

16

[email protected] (C Yao)

AC C

15

SC

7

College of chemistry and molecular engineering, Nanjing Tech University, Nanjing, 211816, P. R.

ACCEPTED MANUSCRIPT 1

Abstract

2

This

work

reports

a

novel

signal

amplification

strategy

based

on

three-dimensional ordered macroporous C60-Poly(3,4-ethylenedioxythiophene)-1-

4

butyl-3-methylimidazolium hexafluorophosphate (3DOM C60-PEDOT-[BMIm][BF6])

5

for ultrasensitive detection of thrombin by cascade catalysis of Au-PEDOT@SiO2

6

microspheres and alcohol dehydrogenase (ADH). Au-PEDOT@SiO2 microspheres

7

were constructed not only as nanocarriers for anchoring the large amounts of

8

secondary thrombin aptamers but also as nanocatalysts to catalyze the oxidation of

9

ethanol efficiently. Significantly, the electrochemical signal was greatly enhanced

10

based on cascade catalysis: Firstly, ADH catalyzed the oxidation of ethanol to

11

acetaldehyde with the concomitant generation of NADH in the presence of NAD+.

12

Then, AuNPs as nanocatalysts could effectively catalyze NADH to produce NAD+

13

with the help of PEDOT as redox probe. Under the optimal conditions, the proposed

14

aptasensor exhibits a linear range of 2.0×10−13-2×10−8 M with a low detection limit of

15

2×10−14 M for thrombin (TB) detection and shows high sensitivity and good

16

specificity.

17

Keywords: 3DOM, C60-PEDOT-[BMIm][BF6], Au-PEDOT@SiO2, aptasensor, TB

18

Introduction

TE D

M AN U

SC

RI PT

3

Thrombin (TB), which is a specific serine protease involved in the coagulation

20

cascade, thrombosis, and hemostasis, has attracted much interest because of its

21

superior functions such as converting soluble fibrinogen into insoluble strands of

22

fibrin and catalyzing many other coagulation-related reactions [1]. It played

23

significant roles in many life processes and relates to a multitude of diseases [2, 3].

24

Therefore, sensitive determination of TB is very important in clinical research and

25

diagnosis. Recently, aptamer-based assays such as chemiluminescence [4] surface

26

plasmon resonance [5] and fluorescence [6] have been developed for detection of

27

thrombin. However, most of these strategies suffer from complex operation, high cost,

28

large-scale and expensive apparatus. Comparatively, aptamer-based electrochemical

29

method shows significant superiority including portability, high sensitivity, simplicity

30

and low-cost instrumentation.

AC C

EP

19

ACCEPTED MANUSCRIPT 1

Aptamers are single-stranded DNA or RNA sequences screened through the process known as SELEX (systematic evolution of ligands by exponential enrichment)

3

from random DNA or RNA libraries [7]. Aptamer based sensors have been broadly

4

used in detection of cancer cells, mental ion, and a variety of proteins [8]. Among

5

these aptasensors, electrochemical aptasensors are the most attractive due to their

6

advantages of fast response, portability, high sensitivity, simple instrumentation, low

7

cost [9-12]. In order to enhance the sensitivity and selectivity of the electrochemical

8

aptasensors, a variety of materials have been employed to modify electrode.

RI PT

2

The conducting polymers show interesting conductivities in the doped state and a

10

variety of optoelectronic and redox properties. EDOT has attracted a great deal of

11

attention as a counter electrode due to its high conductivity, catalytic activity, low cost,

12

ease of synthesis and environmental stability [13-15]. Ionic liquids (ILs) have been

13

also employed as suitable media for EDOT polymerization[16]. ILs act

14

simultaneously as the solvent and supporting electrolyte and their characteristics

15

affect the rapidity of the electropolymerization process as well as the morphology and

16

conductivity of the resulting polymer. In the past, conducting polymer composites

17

encompassing carbon nanotubes (CNTs) or quantum dots have been synthesized, as a

18

blend of enhanced mechanical, surface, thermal, and electrical properties can be

19

achieved due to the synergistic effects of the polymer and the nanomoiety [17-20].

20

Since the discovery of fullerenes in 1985, buckminsterfullerene (C60) has fascinated a

21

large number of scientists due to its remarkable electrochemical properties [21-23].

22

The electrochemical behavior of fullerene films in aqueous solutions has been

23

investigated by many researchers. On the other hand, C60 molecule with conjugate π

24

electron structure, likes olefin molecules, has certain advantages over CNTs.

M AN U

TE D

EP

AC C

25

SC

9

On the other hand, functional nanospheres have increasingly attracted interest due

26

to their potential applications in catalysis, controlled delivery, energy storage,

27

biomedicines. Various solid supports have been used for immobilizing the metal

28

nanoparticles to enhance their catalytic activity and stability. Among them, silica,

29

especially SiO2 spheres, have become promising candidates for supports due to their

30

large loading capacity and numerous surface Si–OH groups. Spheres equipped with

ACCEPTED MANUSCRIPT functional microparticles in their cavities have been developed by some groups. Xia

2

and co-workers synthesized polymer spheres whose interiors were functionalized with

3

movable gold microparticles, which is a novel functional organic sphere structure [24,

4

25]. Yang et al. designed a uniform Ni/SiO2@Au magnetic microspheres: rational

5

design and excellent catalytic performance in 4-nitrophenol reduction [26].

RI PT

1

In this work, we reported a sensitive sandwich-type electrochemical assay for

7

thrombin detection by utilizing electrocatalysis of highly loaded Au-PEDOT@SiO2

8

microspheres-ADH as labels and 3DOM C60-PEDOT-[BMIm][BF6] on the gold

9

electrode as linking thrombin binding aptamer (TBA). To the best of our knowledge,

10

reports on electrochemistry of 3DOM C60-PEDOT-[BMIm][BF6] films are rare. Here,

11

we report the electrochemistry synthesis of C60-PEDOT-[BMIm][BF6] nanosheet

12

films. ADH- MPTS was used as a model enzyme that could be simply immobilized on

13

Au-PEDOT@SiO2 microspheres, which exhibited excellent amperometric response to

14

ethanol, holding great promise to fabricate biosensors for practical applications.

15

Experiments

16

Reagents and material Fullerene

C60

M AN U

TE D

17

SC

6

was

obtained

from

XF

NaNo,

INC,

China.

18

1-butyl-3-methylimidazolium hexafluorophosphate ([BMIm][BF6], purity>99%) was

19

purchased from Lanzhou

20

Sciences (China) and was dried in vacuum at 60

21

desiccator as well. EDOT (aladdin) was used. Chloroauric acid (HAuCl4•4H2O) were

22

purchased from Nanjing Chemical Reagent Co. Ltd (China). Au substrates were

23

provided by Shanghai Institute of Microsystem and Information Technology, Chinese

24

Academy of Sciences (China). They were prepared by sputtering a 200 nm thick Au

25

top layer onto the quartz wafers, which was previously coated with a few nanometers

26

of Cr adhesion layer under vacuum. 6-mercaptohex anol (MCH), ß-nicotinamide

27

adenine

28

MPTS(Trimethoxysilylpropanethiol), Hemoglobin (Hb), TB, bovine serum albumin

29

(BSA), L-cysteine (L-cys) were purchased from Shanghai baoman Co. Ltd (China).

30

Thrombin binding aptamer (TBA) was purchased from by Shanghai Sangon

for 24 h before use, stored in a

AC C

EP

Institute of Chemical Physics, Chinese Academy of

dinucleotide

hydrate

(NAD+),

alcohol

dehydrogenase

(ADH),

ACCEPTED MANUSCRIPT Biotechnology Co. Ltd. (Shanghai, China) with the sequence as follows:

2

5’-NH2-(CH2)6-GGTTGGTGTGGTTGG-3’. TB binding aptamer buffers involved in

3

this work were composed of 300 mM NaCl and 25 mM tris-acetate (pH 8.0). Cyclic

4

voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were

5

performed in the presence of 2 mM [Fe(CN)6] 3−/4−containing 0.1 M KCl. Double

6

distilled water was used throughout the experiments. The human blood serum samples

7

from different persons were provided by Jiangsu Center for Clinical Laboratory

8

(JSCCL), China.

9

Apparatus

SC

RI PT

1

The structures, surface morphology and elemental composition of the 3DOM

11

C60-PEDOT-[BMIm][BF6] electrode were characterized by Philip-X’Pert X-ray

12

diffractometer taken with a Cu KR X-ray source, fieldemission scanning electron

13

microscopy (FESEM, Hitachi S4800). The morphology and composition of

14

Au-PEDOT@SiO2 microspheres were characterized by transmission electron

15

microscopy (TEM, JEOL JEM-200CX) and by Fourier transform infrared (FT-IR)

16

spectroscopic measurements (Bruker, VECTOR22) using KBr pressed disks. UV–vis

17

spectra were recorded on Agilent UV–vis spectrophotometer. The CV and EIS

18

measurements and amperometric measurements were performed using a CHI660D

19

electrochemical workstation (Shanghai CH Instruments, China). A three-compartment

20

electrochemical cell contained a saturated calomel reference electrode (SCE) against

21

which all potentials were measured in this paper, a platinum wire auxiliary electrode

22

and the 3DOM C60-PEDOT-[BMIm][BF6]

23

working electrode. The biosensor responses were measured as the difference between

24

total and background currents.

25

Preparation of Au-PEDOT@SiO2 microspheres composite

AC C

EP

TE D

M AN U

10

modified Au disk electrode (ϕ=2 mm) as

26

A solution of 15 µL EDOT in 15 µL toluene and 20 mg SiO2 were prepared and

27

ultrasound for 15 min, after which a solution of 16 mg sodium persulfate in 300 µL

28

water was added. The mixture was stirred overnight, and the solid was isolated by

29

centrifugation [27]. The solid was washed with water and ethanol by redispersion/cen

ACCEPTED MANUSCRIPT trifugation cycles and dried at 60

. 10 mg PEDOT@SiO2 were dispersed in 4 mL

2

H2O with 1 mL 3% PDDA aqueous solution and ultrasonication for 3 min. After

3

residual PDDA was removed, the positively charged PEDOT@SiO2 nanocomposite

4

was added to 30 mL Au colloid solution and stirred for 8 h and the supernatant liquor

5

was colorless. The Au-PEDOT@SiO2 nanocomposite was washed with water and

6

ethanol for 3 times and dried under vacuum at 60

7

Preparation of Au-PEDOT@SiO2 hollow microspheres-ADH-TBA

.

RI PT

1

According to the literature [28], MPTS sol–ADH composite was prepared by the

9

encapsulation of ADH into MPTS sol with the three-dimensional (3-D) network,

10

which exhibited tunable porosity and high thermal stability, improving the amount of

11

enzyme loaded into the network and retaining enzyme native properties. Briefly, the

12

synthesis of MPTS sol–ADH composite was as follows: 24 µL of MPTS and 10 µL

13

HCl (0.1 M) were mixed with 2 mL of distilled water, and the resulting mixture was

14

stirred vigorously for 30 min to obtain MPTS sol. Then 0.5 mL of ADH solution (8

15

mg/mL in PBS) was added into 0.5 mL of MPTS sol and stirred for 2–3 min. Finally,

16

the resulting composite was stored at 4

17

µL Au-PEDOT@SiO2 microspheres (1 mg/100 µL) and 10 uL ADH-MPTS were

18

shaked for 16 h. Au-PEDOT@SiO2 microspheres-ADH-TBA was obtained.

19

Fabrication of the sensing interface

M AN U

SC

8

EP

TE D

for further use. 200 µL of 2 µM TBA , 20

Gold electrodes using as working electrode were pretreated with ultrasonic

21

cleaning with acetone, anhydrous ethanol, and distilled water for 15 min, respectively.

22

The vertical deposition technique was used to assemble the silica spheres on the gold

23

substrates, forming close-packed crystals. The electrode area was controlled to be

24

0.25 cm2 (0.5 cm×0.5 cm) by an insulating tape. A solution of 17 µL of [BMIm][BF6]

25

with 0.1 M EDOT and 2 mg C60 in 20 mL of ethanol were ultrasonicated for 20 min

26

prior to use. After immersion in a mixture of C60-PEDOT-[BMIm][BF6] solution,

27

slightly stirring and electrodeposited for 100 s with a constant potential of +1.5 V (vs.

28

SCE). Nitrogen gas was used to deaerate oxygen. An ordered pore array was obtained

29

by dissolving the template in aqueous HF (5 %) for 30 s after electrodeposition. The

30

gold electrode surfaces with C60-PEDOT-[BMIm][BF6] were interacted with a

AC C

20

ACCEPTED MANUSCRIPT 1

solution composed of 2 µM TBA for 12 h at 37

2

were then passivated with 1 mM MCH for 60 min and washed with phosphate buffer

3

(PBS, PH 7.4) for three times, the modified electrode was incubated with TB for 40

4

min.

5

Au-PEDOT@SiO2 Microspheres-ADH-TBA. As shown in Scheme1.

the

resulting

TB-functionalized

surfaces

were

treated

with

EP

6

TE D

M AN U

SC

RI PT

Finally,

in 100% humidity. The surfaces

Scheme 1.The fabrication procedure of aptasensor based on 3DOM C60-PEDOT-

8

[BMIm][BF6] electrode

9

Results and discussion

10 11

AC C

7

Characterizations of SEM and TEM In order to verify the successful assembly of 3DOM C60-PEDOT-[BMIm][BF6]

12

nanocomposite film, the morphologies of the 3DOM C60-PEDOT-[BMIm][BF6]

13

composite on a gold substrate were characterized by SEM (Fig.1A). It was clearly

14

observed that the 3DOM C60-PEDOT-[BMIm][BF6] displayed a three dimensionally

15

ordered macroporous structure with a pore size of 400–500 nm. From the Fig.1B of

16

the SiO2 sample, it could be clearly seen that SiO2 particle was composed of many

ACCEPTED MANUSCRIPT nearly monodisperse spherical particles and its surface was not smooth. SEM image

2

of the the uniform and well-dispersed PEDOT@SiO2 sample, as shown in Fig.1C,

3

indicated that the PEDOT@SiO2 nanocomposite had a smooth PEDOT shell. The

4

AuNPs with a diameter of about 20 nm were immobilized evenly onto the surface of

5

PEDOT@SiO2 nanocomposites as Fig.1D. Fig.1E showed TEM images of the SiO2

6

with an average diameter of 500 nm. The synthesized PEDOT@SiO2 was in Fig.1F,

7

the diameter of PEDOT@SiO2 nanocomposites were increased by ~512 nm,

8

demonstrated that the PEDOT shell was ~12 nm thick. Fig.1G showed the TEM

9

image of Au-Fe3O4@SPAN nanocomposites, and it was clear that the composite

10

particles were constituted of three distinct components: a black core of Fe3O4, a gray

11

shell of PEDOT and an outermost layer of many tiny Au.

12

AC C

EP

TE D

M AN U

SC

RI PT

1

13

Fig.1 SEM images of 3DOM C60-PEDOT-[BMIm][BF6](A); SiO2(B); PEDOT@SiO2

14

(C), Au-PEDOT@SiO2(D);TEM of SiO2(E); PEDOT@SiO2(F), Au-PEDOT@ SiO2

15

(H), Au-PEDOT@SiO2 hollow microspheres (G)

16

Electrochemical characterization of the aptasensor

17

CV measurements were used to characterize the electrochemical biosensor, the

18

corresponding results are shown in Fig.2A. The redox probe Fe(CN)64−/3− revealed a

19

reversible CV at the 3DOM C60-PEDOT-[BMIm][BF6] film electrode (curve a). After

ACCEPTED MANUSCRIPT TBA molecules were first self-assembled on the 3DOM C60-PEDOT-[BMIm][BF6]

2

film electrode, the peak current decreased greatly (curve b) due to the electrode

3

surface coated with a negatively charged layer which acts as a barrier preventing

4

ferricyanide anions from approaching the electrode surface. When the negative

5

charged MCH was assembled on TBA-modified gold electrode, the redox current

6

decreased obviously (curve c). After the aptasensor was incubated with TB, the CV

7

response was further decreased. The reason for this was that the inert property of

8

protein thrombin retards the electron transfer tunnel (curve d). Finally, the resulting

9

aptasensor was incubated with Au-PEDOT@SiO2 microspheres-TBA bioconjugate,

10

and the CV response continued to decrease (curve e). Moreover, EIS is a powerful

11

tool for studying the interface properties of surface-modified electrode and the

12

electron-transfer resistance at the electrode surface, as shown in Fig. 2B. In the

13

Nyquist plots, the semicircle diameter of EIS is equal to Ret. The 3DOM C60-PEDOT-

14

[BMIm][BF6] electrode exhibited a very small semicircle (curve a), suggesting a low

15

electron-transfer resistance. When TBA was immobilized on the electrode surface, the

16

Ret increased (curve b), which was ascribed to the inhibition effect of the TBA

17

biomacromolecules for electron transfer. After the successive assembling of MCH, TB

18

and 3DOM C60-PEDOT-[BMIm][BF6] bioconjugate, the resistances were increased

19

with a stepwise increase of semicircle diameter ( curves c, d and e, respectively). The

20

reason for this was that the formed layer acted as the inert electron and mass-transfer

21

blocking layer, and significantly hindered the diffusion of ferricyanide toward the

22

electrode surface. So, the EIS results were in accordance with those of CV

23

characterization.

24

AC C

EP

TE D

M AN U

SC

RI PT

1

ACCEPTED MANUSCRIPT 1

Fig.2 (A) The CV and (B) EIS of 3DOM C60-PEDOT-[BMIm][BF6](a); TBA(b);

2

MCH(c); TB(d) Au-PEDOT@SiO2 hollow microspheres-TBA(e)

3

Characterization of FT-IR Fig.3 shows the FT-IR spectra of SiO2 (curve a), PEDOT@SiO2 nanocomposites

5

(curve b) and Au-PEDOT@SiO2 (curve c) samples. Curve a shows the typical FT -IR

6

spectrum of SiO2, there is a new strong band around 1093 cm−1 originates from the

7

Si–O bond of silica. Moreover, PEDOT-SiO2 nanocomposites (curve b) and

8

Au-PEDOT-SiO2 (curve c) were prepared. Because AuNPs do not have absorption in

9

the infrared region, the characteristic peak of Au-PEDOT-SiO2 sample was almost the

SC

same as that of PEDOT-SiO2 nanocomposites.

TE D

M AN U

10

RI PT

4

11

Fig.3 FT-IR of SiO2 (a); PEDOT@SiO2 (b); Au-PEDOT@SiO2(c);

13

Optimization of experimental conditions for aptasensor

14

The TBA incubation time

EP

12

The incubation time is an important parameter for formation of the G-quarter

16

structure of the aptamer, which is preferred for binding of TB. Fig.4A shows the effect

17

of different incubation times on the current of the aptasensor.The signal output

18

decreased gradually along with increasing in incubation time in the presence of 2 mM

19

[Fe(CN)6]

20

incubation time did not improve the response of the aptasensor. Therefore, 40 min

21

were chosen as the reaction of the TBA.

22

The concentrations of TBA

23

AC C

15

3−/4−

containing 0.1 M KCl, reached a plateau after 40 min. Longer

The concentrations of TBA can affect the response current of this kind of the

ACCEPTED MANUSCRIPT biosensor. Fig.4B showed the response curves of the modified biosensor with different

2

concentrations of TBA. With the increase of the concentrations of TBA, the response

3

current decreased gradually in the presence of 2 mM [Fe(CN)6] 3−/4−containing 0.1 M

4

KCl, reached a constant level within 2.0 µM and remained constant thereafter. Further

5

increment of the TBA would be a waste of this expensive reagent, the concentrations

6

of TBA was set as 2.0 µM in the following experiments.

7

The concentrations of NAD+

RI PT

1

The concentration of NAD+ in electrolytic cell played a key role for the

9

bioelectrocatalytic effect. Therefore, before testing the target TB, we first studied

10

the effect of NAD+ concentration. The aptasensor after incubation of 0.2 nM TB

11

was investigated in PBS (pH 7.0) under successive injection of NAD+. As could be

12

seen from Fig.4C, the responded absolute current values increased with the increase

13

of the concentration of NAD+, and the current tended to reach at saturation when the

14

concentration of NAD+ was higher than 20 µM. Thus, 20 µM NAD+ in PBS was

15

selected in subsequent experiments.

16

The concentrations of ethanol

TE D

M AN U

SC

8

The concentrations of ethanol was also investigated in the range of 0.5-2.5 mM

18

by DPV in the presence of 0.2 nM TB at Fig.4D. With the increase of the

19

concentrations of ethanol, the response current increased gradually within 2.0 mM and

20

kept stable thereafter. The results indicated that 2.0 mM was fully enough for

21

catalysis.

AC C

EP

17

SC

RI PT

ACCEPTED MANUSCRIPT

M AN U

1 2

Fig.4 Dependence of amperometric response of 0.2 nM TB on C60-PEDOT-[BMIm

3

BF6] film electrode, the TBA incubation time (A), the concentrations of TBA (B),

4

NAD+ (C), ethanol (D).

5

Calibration curves of aptasensors

Under the optimal assay conditions, the quantitative analysis of the present

7

biosensor was evaluated by detecting TB in standard solutions using the DPV

8

technique. The response curve of the proposed aptasensor for TB showed a good

9

linear relationship between the reduction peak currents and the analyte concentrations.,

10

the digital reading of the current increases accordingly in the range of

11

2.0×10−13-2×10−8 M, with a limit of detection of 2.0×10-14 M. The regression equation

12

was ip=43.3+3.3log (CTB), with the relative coefficient of 0.982. Such low detection

13

limit was primarily due to the significant signal amplification by cascade catalysis of

14

Au-PEDOT@SiO2 microspheres, alcohol dehydrogenase (ADH) and NAD+. In

15

addition, the analytical performance of the developed aptasensor for TB detection has

16

been compared with those of other similar aptasensors reported. The results were

17

summarized in Table 1. From Table 1, we could see that our proposed aptasensor

18

exhibited a much higher sensitivity and wider linear range, which provided a vigorous

19

evidence of our strategy for highly sensitive detection of TB.

AC C

EP

TE D

6

RI PT

ACCEPTED MANUSCRIPT

1

Fig.5 (A) Amperometric response of 3DOM C60-PEDOT-[BMIm][BF6] electrode in

3

the addition of different concentration TB into 0.1 M pH 7.0 PBS. (B) calibration

4

curve.

5

Table 1 Comparisons of proposed aptasensor with other sandwich-type electrochemi

6

cal aptasensor for thrombin detection. Electrode materials

Detection limit

SWNT/ GO/Au

2 pM

AuNPs

Ferrocenylhexanethiol

Au-PEDOT@SiO2

0.2 nM

0.8-15

[30]

0.34 nM

0.001-50

[31]

0.06 nM

0.1-5

[32]

0.0002-20

This work

0.02 pM

Specificity, reproducibility and stability of the aptasensor

AC C

8

Refs. [29]

EP

@ SiO2

Linear range(nM) 0.01-50

TE D

CoPt/GO

7

M AN U

SC

2

To evaluate the specificity of the electrochemical aptasensor, we challenged the

9

system with other possible interferences, and the results are exhibited in Fig.7. A

10

significant increase induced by the interaction of the aptamer probe with 0.2 nM TB

11

was observed compared to 0.2 nM BSA, 0.2 nM Hb and 0.2 nM L-cys, which

12

indicated indicating the high specificity of the proposed aptasensor for TB detection.

13

The reproducibility of the aptasensor played a critical role in analyzing biological

14

samples in situ without separation. The reproducibility of the aptasensor was

15

investigated by the following methods: the six equally prepared electrodes were

ACCEPTED MANUSCRIPT 1

employed to detect thrombin (0.2 nM) under the same conditions. All electrodes

2

exhibited similar electrochemical responses and a relative standard deviation (RSD)

3

of 4.8 % was obtained. The experimental results suggested the acceptable

4

reproducibility of the proposed aptasensor. The stability of the aptasensor was

5

evaluated every 2 days for long-term storage at 4

6

current response after a storage period of 20 days, which suggested the apatasensor

7

could be used for TB analysis with acceptable stability.

M AN U

SC

RI PT

. It retained 93.1 % of its initial

TE D

8

Fig.6 Specificity of the aptasensor with 0.2 nM BSA, 0.2 nM Hb, 0.2 nM L-cys, 0.2

10

nM thrombin and mixture with 0.2 nM thrombin when 0.2 nM BSA, 0.2 nM L-cys

11

and 0.2 nM Hb were coexisted.

12

Analytical application of the aptasensor

EP

9

TB exists in the form of thrombinogen in healthy human serum samples. During

14

this process, a series of samples were prepared by adding thrombin of different

15

concentrations into 10 fold-diluted healthy human serum samples to examine the

16

feasibility of the proposed strategy. As shown in Table 2, the recovery was acceptable

17

between 79.41 % and 104.7 %. These results indicated that the proposed

18

electrochemical aptasensor provided a potential application in real biological samples.

19

Table 2 Determination of TB added in human blood serum (n=5) with the proposed

20

aptasensor

AC C

13

Sample

Added thrombin/nM

Found thrombin/nM

Recovery/%

ACCEPTED MANUSCRIPT 0.015

0.01602

96.30

2

0.15

0.1456

104.7

3

10.00

10.29

102.7

4

20.00

18.93

79.41

5

30

31.21

86.4

RI PT

1

1 2

Conclusion

In summary, we developed a novel electrochemistry aptasensor for thrombin

4

determination based on Au-PEDOT@SiO2 microspheres-ADH as signal-amplified

5

strategy. The greatly enhanced sensitivity relies upon a mulriple signal amplification

6

scheme: Firstly, the integration of Au-PEDOT@SiO2 microspheres and ADH

7

exhibited

8

electrocatalytic activity. The PEDOT offered the detectable electrochemical signal and

9

further enhanced the electrochemical. Secondly, the corporate electrocatalytic effect of

10

NDA+ and ADH resulted in great signal amplification for the reduction of C2H5OH.

11

Meanwhile, this assay is also proved to be able to distinguish the target TB from the

12

interferents. These unique features endowed the electrochemical aptasensor with high

13

sensitivity for the detection of TB.

14

Acknowledgements

M AN U

biocompatibility

especially

excellent

electroactivity

and

TE D

good

EP

15

SC

3

We greatly appreciate the support from Scientific research and innovation project of ordinary higher education graduate of Jiangsu Province (CXZZ130461).

17

References

18

[1] X.X. Jiao, J.R. Chen, X.Y. Zhang, H.Q. Luo, N.B. Li, A chronocoulometric

19

aptasensor based on gold nanoparticles as a signal amplification strategy for

20 21 22 23 24

AC C

16

detection of thrombin, Anal. Biochem. 441 (2013) 95–100. [2] K. Ishii, L. Hein, B. Kobilkall, S.R. Coughlin, Kinetics of thrombin receptor cleavage on intact cells, J. Biol. Chem. 268(1993)9780–9786. [3] M.A. Shuman, Ann. N.Y. Thrombin-cellular interactions, Acad. Sci. 486(1986) 228–239.

ACCEPTED MANUSCRIPT 1

[4] Y. Huang, S.L. Zhao, Z.F. Chen, M. Shi, J. Chen, H. Liang, An amplified

2

chemiluminescence aptasensor based on bi-resonance energy transfer on gold

3

nanoparticles and exonuclease III-catalyzed target recycling, Chem. Commun.

4

48(2012)11877–11879. [5] M.J. Kwon, J. Lee, A.W. Wark, H.J. Lee, Nanoparticle-enhanced surface plasmon

6

resonance detection of proteins at attomolar concentrations: comparing different

7

nanoparticle shapes and sizes, Anal. Chem. 84(2012)1702–1707.

RI PT

5

[6] L.Y. Xue, X.M. Zhou, D. Xing, Highly sensitive protein detection based

9

on aptamer probe and isothermal nickingenzyme assisted fluorescence signal

11 12

amplification, Chem. Commun. 46(2010)7373–7375.

[7] C. Tuerk, L. Gold, High affinity RNA ligands to human nerve growth factor were

M AN U

10

SC

8

selected from pools of random RNA using SELEX, Science 249(1990)505-510.

13

[8] S.B Xie, Y.Q Chai, R. Yuan, L.J Bai, Y.L. Yuan, Y. Wang, A dual-amplification

14

aptasensor for highly sensitive detection of thrombin based on the

15

functionalized

16

hemin/G-quadruplex, Anal. Chim. Acta, 755 (2012) 46– 53.

18

nanoparticles

composites

and

the

TE D

17

graphene-Pd

[9] Z.B. Chen, L.D. Li, Y.Tian, X.J. Mu, L. Guo, Signal amplification architecture for electrochemical aptasensor. Biosens. Bioelectron 38(2012)37–42. [10] W. Cheng, S.J. Ding, Q. Li, T.X. Yu, Y.B. Yin, H.X. Ju, G.S. Ren, A simple

20

electrochemical aptasensor for ultrasensitive protein detection, Biosens.

21

Bioelectron. 36(2012)12–17.

23

[11] T. Hianik, J. Wang, Electrochemical aptasensors—recent achievements and

AC C

22

EP

19

perspectives, Electroanalysis 21 (2009) 1223–1235.

24

[12]Z.S. Wu, H. Zhou, S.B. Zhang, G.L. Shen, R.Q. Yu, Electrochemical aptameric

25

recognition system for a sensitive protein assay based on specific target

26

binding-induced rolling circle amplification, Anal. Chem. 82 (2010) 2282–2289.

27

[13] R. Kawano, T. Katakabe, H. Shimosawa, M.K. Nazeeruddin, M. Gratzel, H.

28

Matsui, T. Kitamura, N. Tanabe, M. Watanabe, Phys. Solid-state dye-sensitized

29

solar cells using polymerized ionic liquid electrolyte and platinum-free counter,

30

Chem. Chem. Phys. 12(2010)1916-1921.

ACCEPTED MANUSCRIPT 1

[14] K. Manseki, W. Jarernboon, Y. Youhai, K. J. Jiang, K. Suzuki, N. Masaki, Y. Kim,

2

J.Xia, S. Yanagida, Solid-state dye-sensitized solar cells fabricated by coupling

3

photoelectrochemically deposited poly(3,4-ethylenedioxythiophene) (PEDOT)

4

with silver-paint on cathode, Chem. Commun. 47(2011)3120-3122. [15] W. Hong, Y. Xu, G. Lu, C. Li, G. Shi, Transparent graphene/PEDOT–PSS

6

composite films as counter electrodes of dye-sensitized solar cells, Electrochem.

7

Commun. 10(2008)1555-1558.

RI PT

5

[16] K. Wagner, J.M. Pringle, S.B. Hall, M. Forsyth, D.R. MacFarlane, D.L. Officer,

9

Investigation of the electropolymerisation of EDOT in ionic liquids, Synth. Met.

10

SC

8

153(2005)257-260.

[17] Y. K. Zhou, B. L. He, W. J. Zhou, J. Huang, X. H. Li, B. Wu, H. L. Li,

12

Electrochemical capacitance of well-coated single-walled carbon nanotube with

13

polyaniline composites, Electrochim. Acta 49(2004)257–262.

M AN U

11

[18] H. Zengin, W. Zhou, J. Jin, R. Czerw, D. W. Smith, L. Echegoyen, D. L. Carroll,

15

S. H. Foulger, Ballato, Canbon nanotube doped polyaniline, J. Adv. Mater.

16

14(2002)1480–1483.

18 19

[19] Y. Saito, S. Uemura, K.Jpn. Hamaguchi, Cathode ray tube lighting elements with carbon nanotube field emitters, J. Appl. Phys. 37(1998) L346–L348. [20] S. Bhandari, M. Deepa, A. K. Srivastava, C. Lal, R. Macromol. Kant, Poly (3,4-

EP

17

TE D

14

ethylenedioxythiophene)(PEDOT)-coated MWCNTs tethered to conducting

21

substrates: facile electrochemistry and enhanced coloring efficiency, Rapid

22

Commun. 29(2008) 1959–1964.

AC C

20

23

[21] A. D. Bettencourt-Dias, K. Winkler, W. R. Fawcett, A. L. Balch, The influence of

24

electroactive solutes on the properties of electrochemically formed fullerene

25

C60-based films, J. Electroanal. Chem. 549(2003) 109-117.

26

[22] R.N. Goyal, V. K. Gupta, N. Bachheti, R.A. Sharma, Electrochemical sensor for

27

the determination of dopamine in presence of high concentration of ascorbic acid

28

using a fullerene-C60 coated gold electrode, Electroanalysis, 20(2008)757-764.

29

[23] R.N. Goyal, V.K. Gupta, N. Bachheti, Fullerene-C60-modified electrode as a

30

sensitive voltammetric sensor for detection of nandrolone—An anabolic steroid

ACCEPTED MANUSCRIPT 1

used in doping, Anal. Chim. Acta, 597(2007)82-89.

2

[24] M.A. Rahman, J.I. Son, M.S. Won, Y.B. Shim, Gold nanoparticles doped

3

conducting polymer nanorod electrodes: ferrocene catalyzed aptamer-based

4

thrombin immunosensor, Anal. Chem. 81 (2009) 6604–6611 [25] K.Kamata, Y. Lu, Y.J. Xia, Synthesis and characterization of monodispersed

6

core-shell spherical colloids with movable cores, J. Am. Chem. Soc.

7

125( 2003)2384-2385.

RI PT

5

[26] S.H. Zhang, S.L Gai, F. He, Y.L Dai, P. Gao, L. Li, Y.J Chen and P.P Yang,

9

Ni/SiO2@Au magnetic hollow microspheres: rationaldesign and excellent

10

SC

8

catalytic performance in 4-nitrophenol reduction, Nanoscale 6(2014)7025–7032 [27] T. L. Kelly, P. Y. C. Sara, Y. Yamada, K. Yano, M. O. Wolf, Influence of surface

12

morphology on the colloidal and electronic behavior of conjugated

13

polymer-silica microspheres, Langmuir, 24(2008) 9809-9815.

M AN U

11

[28] B.K. Jena, C.R. Raj, Electrochemical biosensor based on integrated assembly of

15

dehydrogenase enzymes and gold nanoparticles, Anal.Chem.78(2006) 6332–

16

6339.

TE D

14

[29] Y.L. Yuan, X.X. Gou, R. Yuan, Y.Q. Chai, Y. Zhuo, L. Mao, X.X. Gan,

18

Electrochemical aptasensor based on the dual- amplifycation of G-quadruplex

19

horseradish peroxidase-mimicking DNAzyme and blocking reagent-horseradish

20

peroxidase, Biosens. Bioelectron. 26(2011)4236–4240.

EP

17

[30] J.H. Chen, J. Zhang, J. Li, H.H. Yang, F.F. Fu, G.N. Chen, An ultrasensitive

22

signal-on electrochemical aptasensor via target-induced conjunction of split

23

AC C

21

aptamer fragments, Biosens. Bioelectron. 25(2010)996–1001.

24

[31] Y. Wang, Y. Ruo, Y.Q. Chai, Y.L. Yuan, L.J. Bai, Y.H. Liao, A multi-amplification

25

aptasensor for highly sensitive detection of thrombin based on high-quality

26

hollow CoPt nanoparticles decorated graphene, Biosens.Bioelectron. 30(2011)

27

61–66.

28

[32] Y.H.Wang, X.X. He, K.M. Wang, X.Q. Ni, J. Su, Z.F. Chen, Electrochemical

29

detection of thrombin based on aptamer and ferrocenylhexanethiol loaded silica

30

nanocapsules, Biosens.Bioelectron. 26(2011)3536–3541.