Molecularly Imprinted Polymer

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Novel hybrid structure silica/CdTe/molecularly imprinted polymer: synthesis, specific recognition and quantitative fluorescence detection of bovine hemoglobin Dong-Yan Li, Xi-Wen He, Yang Chen, Wen-You Li, and Yu-Kui Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 20 Nov 2013 Downloaded from http://pubs.acs.org on November 25, 2013

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ACS Applied Materials & Interfaces

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Novel hybrid structure silica/CdTe/molecularly imprinted polymer:

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synthesis, specific recognition and quantitative fluorescence detection

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of bovine hemoglobin

4 5

Dong-Yan Li,a,b Xi-Wen He,a,b Yang Chen,a,b Wen-You Li,*,a,b Yu-Kui Zhanga,b,c

6 7

a

8

University, Tianjin 300071, P. R. China

9

b

State Key Laboratory of Medicinal Chemical Biology, and Department of Chemistry, Nankai

Synergetic Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, P.

10

R. China

11

c

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Chinese Academy of Sciences, Dalian 116011, P. R. China

National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics,

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ACS Paragon Plus Environment


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ABSTRACT：：This work presented a novel strategy for the synthesis of the hybrid

2

structure silica/CdTe/molecularly imprinted polymer (Si-NP/CdTe/MIP) to recognize

3

and detect the template bovine hemoglobin (BHb). Firstly, amino-functionalized silica

4

nanoparticles (Si-NP) and carboxyl-terminated CdTe quantum dots (QDs) were

5

assembled

6

(1-(3-dimethylaminopropyl)-3- ethylcarbo diimide hydrochloride) chemistry. Next,

7

Si-NP/CdTe/MIP was synthesized by anchoring molecularly imprinted polymer (MIP)

8

layer on the surface of Si-NP/CdTe through the sol-gel technique and surface

9

imprinting technique. The hybrid structure possessed the selectivity of molecular

10

imprinting technique and the sensitivity of CdTe QDs as well as well-defined

11

morphology. The binding experiment and fluorescence method demonstrated its

12

special recognition performance towards the template BHb. Under the optimized

13

conditions, the fluorescence intensity of the Si-NP/CdTe/MIP decreased linearly with

14

the increase of BHb in the concentration range of 0.02-2.1 µM, and the detection limit

15

was 9.4 nM. Moreover, the reusability and reproducibility, and the successful

16

applications in practical samples indicated the synthesis of Si-NP/CdTe/MIP provided

17

an alternative solution for special recognition and determination of protein from real

18

samples.

into

composite

nanoparticles

(Si-NP/CdTe)

using

the

EDC

19 20

KEYWORDS: molecularly imprinted polymer, quantum dots, fluorescent detection,

21

protein recognition 2


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1. Introduction

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Molecular imprinting is a well-known technique for the creation of tailor-made

3

binding sites with memory of the shape, size and functional groups of the template.1

4

Synthesizing molecularly imprinted polymer (MIP) involves the copolymerization of

5

functional monomers and cross-linkers in the presence of the template, and the special

6

tailor-made binding sites are exposed after the removal of the template. Thus, MIP has

7

two characteristics of the most important features: the ability of recognition and

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binding special target molecule.2 Recently, MIP has become very attractive and been

9

applied in the fields of chem/biosensor3,4, extraction/separation5, catalysis6, antibody

10

mimetics7 and drug delivery8 owing to its desired selectivity, physical robustness,

11

good stability as well as low cost and reusability.1

12

Although the synthesis of MIP towards small molecules is straightforward now, the

13

imprinting of biomacromolecules like proteins continues to be a significant

14

challenging.9,10 The difficulties faced by proteins for imprinting applications lie in the

15

large molecular size, diffusion limitation, complexity, flexible conformation and

16

solubility.11 To overcome these problems, surface imprinting technique has been

17

adopted to various support materials (e.g., silica nanoparticles,12 silica nanotubes,13

18

alumina membrane14 and magnetic nanoparticles15). Particularly, silica nanoparticles

19

are popular substrates for imprinted MIP for many reasons: a) the synthesis of silica

20

nanoparticles themselves is a well-established process; b) silica possesses numerous

21

hydroxyl groups on its surface and was employed as grafting sites for the surface 3



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polymer; c) the chemical/mechanical stability, non-toxicity and biocompatibility of

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silica make it an attractive substrate in many fields.16 The introduction of the thin

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imprinted layer coating on the surface of silica nanoparticles via surface imprinting

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enables the elution and rebinding of the target template easily. Meanwhile, the

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selective recognition and determination of proteins in biological samples are of great

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significance in life science. Hence, photoluminescence materials (e.g. QDs) have been

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proposed as feasible strategies for synthesizing MIP.

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QDs are often described as ‘artificial atom’, exhibiting unique luminescence

9

characteristics and electronic properties such as wide and continuous absorption

10

spectra, narrow emission spectra and high light stability.17,18 Therefore, some MIPs

11

have been synthesized by encapsulating QDs in cross-linked polymers combining the

12

selectivity of molecular imprinting technology and the sensitivity of QDs.19,20 For

13

example, Yan’s group has developed a type of MIP-based room-temperature

14

phosphorescence by anchoring MIP layer on the surface of Mn-doped ZnS QDs for

15

detecting pentachlorophenol.20 Wang’s group has introduced a fluorescence

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nano-sensing material by anchoring the MIP layer on 1-vinyl-3-octylimidazolium

17

ionic liquid-modified CdSe/ZnS QDs for optosensing of tocopherol.21 Our group has

18

developed a series of protein-imprinted polymers by encapsulating CdTe QDs into

19

silicon material, in which the synthesis of MIPs is under the condition of water phase

20

and the morphology of MIPs left much to be desired.22-24 The reasons could be

21

explained that because the size of QD is small, some of the imprinted outer layers 4


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would actually be coated on QD aggregates comprised of several QDs and the shape

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of MIP was aggregated.25-27 In order to improve the morphology of MIP and possess a

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favorable fluorescence spectrum simultaneously, Chen’s group proposed a novel

4

approach for creating MIP-capped QDs sensing for imprinting 2,4,6-trinitrotoluene by

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a seed-growth method via a sol-gel process.4 Based on these studies, a novel strategy

6

for synthesizing a unique hybrid structure for imprinting target protein was proposed.

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The Si-NP/CdTe/MIP was constructed by the sol-gel technique and surface molecular

8

imprinting technique, combining the merits of MIP and QDs as well as well-defined

9

morphology. The hybrid structure possessed multifunctional properties for selective

10

recognition, separation and detection of protein from complex samples.

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In this work, carboxyl-terminated CdTe QDs and amino-functionalized silica

12

nanoparticles were firstly assembled into Si-NP/CdTe by covalently linking the highly

13

fluorescent CdTe QDs on the surface of the Si-NP. Numerous “satellites” of QDs were

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linked on the surface of the Si-NP by the way of amide bonding, which owned

15

photoluminescence properties and played the role of well-defined support substrates

16

for nanostructured imprinted materials by surface imprinting technique. And then, the

17

MIP layer was anchored on the surface of Si-NP/CdTe to form the unique

18

Si-NP/CdTe/MIP which possessed the high selectivity of molecular imprinting

19

technology (MIT) and the high sensitivity of CdTe QDs. Besides, the

20

Si-NP/CdTe/MIP provided better site accessibility, lower mass transfer resistance for

21

special recognition and detection performance towards the corresponding template 5



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protein. Moreover, the reusability and reproducibility as well as the successful

2

applications in practical samples indicated the approach for synthesizing

3

Si-NP/CdTe/MIP provided an alternative solution for special recognition and

4

detection of protein.

5 6

2. Experimental Section

7

2.1 Materials

8

All reagents used were of at least analytical grade. Tellurium powder,

9

CdCl2·2.5H2O,

NaBH4, ammonium

10

ethanesulfonic

acid

11

tetraethoxysilicane

12

3-Mercaptopropionic acid (MPA) was purchased from Alfa Aesar. Triton X-100 was

13

purchased from Solarbio Co. Bovine hemoglobin (BHb, pI (isoelectric point) = 6.9,

14

MW (molecular weight) = 64.5 kDa), bovine serum albumin (BSA, pI = 4.9, MW =

15

66.0 kDa), lysozyme (Lyz, pI = 11.0, MW = 14.4 kDa), ovalbumin (OB, pI = 4.7, MW

16

= 45.0 kDa) and 1-(3-dimethylaminopropyl)-3- ethylcarbo diimide hydrochloride

17

(EDC) were purchased from Sigma. Bovine blood was kindly gifted by Xiaochuan

18

Biotech. Co. Ltd. (Tianjin, China). The urine was collected from healthy volunteer.

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2.2 Characterization

(MES),

(TEOS)

hydroxide

(NH3·H2O),

3-aminopropyltriethoxysilane were

obtained

from

J&K

(2-N-morpholino) (APTES)

and

Chemical

Co.

20

Fluorescence (FL) measurements were performed on an F-4500 fluoro-

21

spectrophotometer (Hitachi, Japan). UV-Vis spectra (200-800 nm) were recorded on a 6


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UV-2450 spectrophotometer (Shimadzu, Japan). Fourier-transform infrared (FT-IR)

2

spectra (4000–400 cm-1) in KBr were recorded by using a Vector 22 FT-IR

3

spectrophotometer (Bruker, Germany). The morphologies and sizes of samples were

4

obtained by field-emission scanning electron microscope (FESEM, JSM-7500F, JEOL,

5

Japan) and high-resolution transmission electron microscope (HRTEM, Tecnai G2 F20,

6

Philips, Netherlands). Energy dispersive X-ray (EDS, Oxford, Britain) was used to

7

analyze the chemical elements on the surface of samples. X-ray

8

spectroscopy (XPS) measurements were performed with an

9

spectrometer (Thermo-VG Scientific, USA) with ultra-high vacuum generators. Gel

10

electrophoresis was carried out by sodium dodecyl sulfate polyacrylamide gel

11

electrophoresis (SDS-PAGE) with 18% running gel and 6% stacking gel (Bio-Rad,

12

USA).

13

2.3 Synthesis of Carboxyl-terminated CdTe QDs

photoelectron ESCALAB 250

14

The carboxyl-terminated CdTe QDs were synthesized according to previous reports

15

with some modifications.28 Briefly, after tellurium powder was reduced completely by

16

excessive NaBH4, 1.5 mL of freshly prepared NaHTe aqueous solution was injected

17

into a CdCl2-MPA solution which was deaerated by N2 for 30 min. The molar ratio of

18

Cd2+/HTe-/MPA was set as 1:0.5:2.4. Then the solution was heated up until boiling.

19

After being refluxed for 2 h, the MPA stabilized CdTe QDs were obtained.

20

2.4 Synthesis of Amino-functionalized Si-NP

21

Amino-functionalized silica nanoparticles (Si-NP) were synthesized based on a 7



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modified Stöber method.29,30 50 mL of ethanol and 50 mL of water were added to a

2

250-mL round-bottomed flask with a mechanical stirring bar. 10 mL of NH3·H2O was

3

added and the mixture was stirred at 600 rpm. 5 mL of TEOS was added drop by drop

4

and then the resultant mixture was stirred overnight. 5 mL of APTES was then added

5

and the mixed solution was stirred for additional 12 h. The amino-functionalized silica

6

nanoparticles were centrifuged and washed with ethanol at least for five times before

7

being dried under vacuum.

8

2.5 Fabrication of Si-NP/CdTe

9

10 mg of Si-NP were dispersed in 20 mL of MES buffer (0.1 mmol), meanwhile,

10

EDC (20 mg/mL) were dispersed in CdTe aqueous solution. After the carboxyl groups

11

were activated, CdTe QDs were added drop by drop into the solution of Si-NP.31,32

12

The final mixture was stirred for 12 h at room temperature in the dark. Finally, the

13

Si-NP/CdTe composite nanoparticles were centrifuged at 13 000 rpm for 5 min for

14

three times (unbound QDs were removed) and then washed with phosphate buffer

15

saline (PBS) (0.01 mol/L, pH 7.0).

16

2.6 Preparation of Si-NP/CdTe/MIP

17

The Si-NP/CdTe nanoparticles were dispersed in 20 mL of PBS buffer solution

18

(0.01 mol/L, pH 7.0), in which 80 µL of APTES and 10 mg of BHb were added and

19

stirred for 30 min. And then, 100 µL of TEOS and 100 µL of NH3·H2O were added

20

and stirred for 2h. Finally, the Si-NP/CdTe/MIP nanoparticles which owned specific

21

imprinted cavities were obtained after the removal of embedded template protein BHb. 8


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Briefly, 20 mL 0.5 % Triton X-100 solution was added to the resultant polymers and

2

the mixture was stirred about 1 h at room temperature, and then the polymers were

3

centrifuged at 13 000 rpm for 5 min and washed with 0.5 % Triton X-100 until no

4

template protein was detected by the UV-Vis spectrophotometer at 406 nm. The

5

non-imprinted polymer (Si-NP/CdTe/NIP) was prepared under the same conditions in

6

the absence of the template.

7

2.7 Binding Experiment

8

The adsorption experiment was performed to evaluate the binding capacity of the

9

Si-NP/CdTe/MIP. 30 mg of Si-NP/CdTe/MIP and Si-NP/CdTe/NIP were added to 3

10

mL of 0.2 mg/mL BHb solution (0.01 mol/L PBS, pH 7.0) respectively, and placed on

11

the table concentrator. The supernatant and polymers were separated by centrifugation

12

and detected by the UV-Vis spectrophotometer. The adsorption capacity (Q) is

13

calculated using the equation below:

14

Q = (C0 - Ct) V/ W (mg g-1)

15

C0 and Ct are the initial and equilibrium concentrations of the template protein BHb,

16

respectively, V (mL) is the volume of the initial solution, W (g) is the weight of the

17

Si-NP/CdTe/MIP.

18

2.8 Fluorescence Measurement

(1)

19

In the experiment, all the fluorescence (FL) intensity detections were under the

20

same conditions: the slit widths of the excitation and emission were both 10 nm, and

21

the excitation wavelength was set at 450 nm with a recording emission range of 9



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490-700 nm. The photomultiplier tube voltage was set at 700 V. The appropriate

2

Si-NP/CdTe/MIP and Si-NP/CdTe/NIP solution were respectively added in 5 mL

3

standard colorimetric tube and the given concentration of protein was added

4

successively. The mixture was mixed thoroughly and scanned by the F-4500

5

fluorospectrophotometer.

6

2.9 Bovine Blood and Urine Samples

7

Bovine blood and urine samples were used to demonstrate the applicability of the

8

Si-NP/CdTe/MIP nanomaterial for specific recognition and quantitative fluorescence

9

detection of BHb. Briefly, the bovine blood sample was diluted 300-fold with PBS

10

buffer (0.01 mol/L, pH 7.0), and then 6 mg of Si-NP/CdTe/MIP and Si-NP/CdTe/NIP

11

were immerged with 400 µL of 300-fold dilution of bovine blood, respectively. And

12

then, the Si-NP/CdTe/MIP and Si-NP/CdTe/NIP were washed with PBS buffer (0.01

13

mol/L, pH 7.0) to remove nonspecific binding proteins. Next, 0.5 % Triton X-100 was

14

employed to elute the specifically adsorbed BHb. Finally, 20 µL of each sample was

15

used for SDS-PAGE analysis.

16

100-fold dilution of urine and 6000-fold dilution of bovine blood were used to

17

detect and assess the analysis method and the accuracy of the measurement system.

18

As no BHb in the collected urine was detectable by the method, the recovery was

19

carried out by the standard addition method with 5.0 × 10-7 mol/L to 15.0 × 10-7 mol/L

20

BHb. As well, the detection of BHb in bovine blood sample was performed based on

21

the premise that the concentration of BHb in 6000-fold dilution of bovine blood was 10


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in the linear range, and then the recovery was performed by the spiked BHb.

2 3

3. Results and Discussions

4

3.1 Preparation and Characterization of the Hybrid Structure

5

The Si-NP/CdTe/MIP was prepared via a multistep procedure (Scheme 1). In this

6

protocol, the monodispersed silica nanoparticles were prepared by the Stöber method

7

and subsequently modified with APTES to form the amino-functioned Si-NP.33 MPA

8

on the surface of CdTe QDs acted as a stabilizer and provided carboxyl groups for

9

conjugating with amino-functionalized. Next, the copolymerization of the composited

10

Si-NP/CdTe, APTES (functional monomer), template (BHb) and TEOS (cross-linking

11

agent) formed polymeric network around the template. Finally, the Si-NP/CdTe/MIP

12

which owned specific imprinted cavities were obtained after the removal of embedded

13

template protein BHb.

14 15

(Preferred position for Scheme 1)

16 17

As shown in Figure S1, the size of the CdTe QDs was about 3 nm. The particle size

18

of uniform Si-NP was about 90 nm (Figure 1a). After linking CdTe QDs around the

19

Si-NP surfaces, FESEM analysis showed that the resultant Si-NP/CdTe nanoparticles

20

were highly spherical in shape and uniform in size (Figure 1b), which played the role

21

of well-defined support substrates for nanostructured imprinted materials. Because the 11



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particle size of the CdTe QDs was only about 3 nm (as shown in the Figure S1), no

2

obviously change can be observed from the FESEM image of the Si-NP/CdTe.

3

However, CdTe QDs being linked outside the Si-NP surface can be seen from the

4

HRTEM image of Si-NP/CdTe (Figure 1c). After the MIP layer was anchored on the

5

Si-NP/CdTe, it was observed from Figure 1d that the thin molecularly imprinted layer

6

was about 10 nm in the outmost layer, which provided better site accessibility, lower

7

mass transfer resistance and well-defined material shape for special recognition

8

towards the corresponding template protein.34 The result indicated that the hybrid

9

structure was successfully synthesized, and it owned Si-NP as the support substrates,

10

the CdTe QDs with the fluorescence properties and the thin molecularly imprinted

11

layer for specific recognition of protein.

12 13

(Preferred position for Figure 1)

14 15

In order to further confirm the successful synthesis of the hybrid structure, EDS and

16

XPS spectroscopy analysis were employed. As shown in Figure S2A, the signal of N

17

(K-alpha) at 0.392 keV was appeared, indicating amino group was successfully

18

modified onto the surface of the Si-NP. And then, it can be seen from Figure S2B that

19

the hybrid structure owned the elements of Cd (L-alpha) at 3.124 keV and Te (L-alpha)

20

at 3.769 keV, which confirmed that CdTe QDs were successfully modified on the

21

surface of the Si-NP. The XPS spectrum of the as-prepared hybrid structure was also 12


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recorded to analyze the surface chemical compositions and gain more structure

2

information (Figure S3). In the spectrum, the elements of Si, N, C and O could be

3

clearly seen. The signals of Cd and Te are very weak, which indicated CdTe QDs were

4

embedded in the inside of MIP layer and only a trace amount of them were detected.

5

FT-IR spectra were further investigated and shown in Figure S4. The characteristic

6

signals of the stretching vibration of Si-O-Si at 1087 cm-1 and Si-O vibration at 794

7

cm-1 and amino group of APTES at 3414 cm-1, 1641 cm-1 and 1618 cm-1 were

8

observed in the spectrum of Si-NP (Figure S4b), which proved the amino groups were

9

successfully modified onto the surface of silica nanoparticles. The successful

10

synthesis of Si-NP/CdTe composite nanoparticles were further studied as shown in

11

Figure S4c, which had a peak at 1400 cm-1 due to the vibration of CO-NH, and the

12

peak at 1560 cm-1 of Figure S4a reappeared. Because there is no introduction of new

13

functional groups, compare with Figure S4c, the spectrum of Si-NP/CdTe/MIP had no

14

obvious change and was shown in Figure S4d.

15

3.2 Adsorption Properties of Si-NP/CdTe/MIP

16

3.2.1 Adsorption Capacity

17

To investigate the binding affinity of Si-NP/CdTe/MIP and Si-NP/CdTe/NIP, the

18

adsorption test was performed by adsorbing the template BHb. The adsorption

19

capacity of Si-NP/CdTe/MIP and Si-NP/CdTe/NIP were shown in Figure 2. In this

20

case, the experimental adsorption capacity of the Si-NP/CdTe/MIP was 19.7 mg/g.

21

Different from Si-NP/CdTe/MIP, the Si-NP/CdTe/NIP showed low binding affinity 13



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toward the protein BHb and the adsorption capacity was 5.9 mg/g. The result could be

2

explained that special cavities of BHb were formed during the imprinting process and

3

Si-NP/CdTe/MIP showed higher binding affinity for BHb. On the contrary, as for

4

Si-NP/CdTe/NIP, there was lack of recognition sites and the non-specific adsorption

5

had a dominant effect. Therefore, its adsorption capacity was low.

6 7


8 9

3.2.2 Adsorption-desorption and Reproducibility

10

Desorption and regeneration are important indicators for the application of the

11

Si-NP/CdTe/MIP nanomaterial. The adsorption-desorption cycles were repeated six

12

times using the same batch of Si-NP/CdTe/MIP and Si-NP/CdTe/NIP to study their

13

reversible binding and release behavior. As shown in Figure 3, it could be seen the

14

adsorption capacity of the Si-NP/CdTe/MIP lost about 8.5% of its affinity on average

15

over six cycles and the Si-NP/CdTe/NIP had not obvious change. It is possible that

16

some specific cavities were blocked during the process of adsorption or destroyed

17

after rewashing. On the contrary, the Si-NP/CdTe/NIP had no imprinted recognition

18

site, and the washing was insignificant. The results indicated that Si-NP/CdTe/MIP

19

and Si-NP/CdTe/NIP retained their recovery efficiency, which was a clear superiority

20

over disposable materials and could be used repeatedly at least within six cycles.

21

Reproducibility is also important for the Si-NP/CdTe/MIP as performing material. 14


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The reproducibility experiment was studied by using six different batches of

2

Si-NP/CdTe/MIP and Si-NP/CdTe/NIP prepared at different times. Through three

3

parallel determinations to every batch, the result showed that the reproducibility of

4

Si-NP/CdTe/MIP and Si-NP/CdTe/NIP were satisfactory with RSD less than 5%.

5 6


7 8

3.3 Optosensing of BHb by the Si-NP/CdTe/MIP

9

3.3.1 Influence of Monomer and Crosslinking Agent

10

To synthesize the effective and favorable molecularly imprinted polymer, the

11

amount of functional monomer APTES and cross-linking agent TEOS were

12

investigated (Figure S5). The quenching amount, defined as (F0/F)-1, was used as the

13

index of quenching capacity. As shown in Figure S5a, the FL intensity of

14

Si-NP/CdTe/MIP gradually decreased with increasing the amount of APTES, which is

15

possible that the native structure of CdTe QDs was destroyed by APTES during the

16

process of fabricating the silica shell onto the Si-NP/CdTe composite nanoparticles.

17

According to the quenching amount and the FL intensity of Si-NP/CdTe/MIP, 80 µL

18

APTES was chosen for the appropriate amount for the further experiments. Based on

19

the same principle, 100 µL TEOS was selected as cross-linking agent for performing

20

the fluorescent sensor (Figure S5b).

21

3.3.2 Effect of pH and the Stability 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

The pH plays an important role in determining the structure of sol-gel-derived

2

polymer, and has obvious effects on the activity and the charge of BHb as well as the

3

FL intensity of Si-NP/CdTe/MIP.35 As shown in Figure 4, the effects of pH on the

4

quenching amount of the Si-NP/CdTe/MIP are more obvious than that of the

5

Si-NP/CdTe/NIP. That was because the tailor-made binding sites were formed in

6

Si-NP/CdTe/MIP and pH affects three dimensional structure proteins and hence

7

affects BHb rebinding at different pH. Besides, the synthesis of CdTe QDs was under

8

the alkaline, and the FL intensity was low under the condition of pH less than 7.

9

However, with the increase of pH, the quenching amount of Si-NP/CdTe/MIP

10

decreases quickly because the molecular imprinting silica layer could be ionized at

11

high pH, which would affect the interaction between the template protein and the

12

fluorescent sensor. Above all, BHb may be denatured under high or low pH.

13

Combining the quenching amount of Si-NP/CdTe/MIP and the physiological

14

condition, 0.01 mol/L PBS buffer (pH 7.0) was selected as the optimized binding

15

media for further experiments.

16 17


18 19

Under the optimized conditions, the fluorescence stability was evaluated by the

20

repeated detections of the fluorescence intensity of Si-NP/CdTe/MIP every 10 min.

21

As shown in Figure 5, the change of the FL intensity of Si-NP/CdTe/MIP was not 16


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1

obvious, which showed a stable emission of Si-NP/CdTe/MIP within 60 min. And

2

then, the change of fluorescence intensity during the storage of Si-NP/CdTe/MIP was

3

also investigated (Figure S6). When the sensor was stored for 7 days, the fluorescence

4

intensity retained 91% of its original response, which indicated the unique sensor has

5

acceptable storage stability. These results might demonstrate that the molecularly

6

imprinted layer was effectively anchored on the surface of Si-NP/CdTe and the CdTe

7

QDs were well protected by the molecularly imprinted layer.

8 9


10 11

3.3.3 Si-NP/CdTe/MIP with BHb of Different Concentrations

12

To study the binding affinity properties of Si-NP/CdTe/MIP and Si-NP/CdTe/NIP

13

with the template BHb, the fluorescence analysis was carried out. The fluorescence

14

quenching followed the Stern-Volmer equation.

15

F0 /F=1+KSV[Q]

(2)

16

F0 and F were the FL intensity of CdTe QDs in the absence and presence of the

17

template, respectively, KSV was the Stern-Volmer constant, [Q] was the concentration

18

of BHb. The ratio of KSV,MIP to KSV,NIP was defined as the imprinting factor (IF).

19

As shown in Figure 6, the FL intensity of the Si-NP/CdTe/MIP and

20

Si-NP/CdTe/NIP decreased linearly with the increase of the concentrations of BHb. It

21

was seen that the decrease of FL intensity of the Si-NP/CdTe/MIP was much larger 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

than that of Si-NP/CdTe/NIP at the same concentration of BHb, which was because

2

specific molecular recognition sites of predetermined selectivity were formed in the

3

Si-NP/CdTe/MIP; however, the Si-NP/CdTe/NIP had no imprinting cavity. Under the

4

optimized conditions, the fluorescence intensity of the Si-NP/CdTe/MIP decreased

5

linearly with the increase of the template protein BHb in the concentration range of

6

0.02-2.1 µM, and the detection limit was 9.4 nM. Based on the quenching amount of

7

the FL intensity of Si-NP/CdTe/MIP with BHb, the Si-NP/CdTe/MIP as a fluorescent

8

sensor was used to detect template protein. The precision for five replicate detections

9

of BHb at 0.20 µM was 0.98% (relative standard deviation) and the imprinting factor

10

(IF) was 3.79, which indicates that the Si-NP/CdTe/MIP can recognize the template

11

BHb.

12 13


14 15

3.3.4 Selective and Competitive Binding

16

The fluorescent response of Si-NP/CdTe/MIP towards various proteins was

17

performed to study its selectivity towards the template. As shown in Figure 7a,

18

Si-NP/CdTe/MIP had strong response to BHb which caused significant change of FL

19

intensity with high quenching amount, more obvious than other proteins. In the

20

process of synthesis of the Si-NP/CdTe/MIP, many specific imprinted cavities with

21

the memory of the shape, size and functional groups of BHb were generated. 18


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1

Although Lyz and OB were small to get into the cavities, the specific recognition sites

2

were not complementary to Lyz and OB, thus they are not easy to quench the

3

fluorescence of the Si-NP/CdTe/MIP. As for BSA, although its molecular weight was

4

similar with BHb, it could not be completely matched with the preparative imprinted

5

sites of BHb. Therefore, BSA also could not effectively quench the fluorescence of

6

the Si-NP/CdTe/MIP.

7

As well, the competitive binding experiments were performed by changing the ratio

8

of different concentrations of BSA and the fixed concentration of BHb. As shown in

9

Figure 7b, the FL intensity of Si-NP/CdTe/MIP had no significant change with the

10

increase of CBSA/CBHb ratio. The result can be explained that although BSA has the

11

similar molecular weight with BHb, BHb is a tetrameric protein composed of pairs of

12

two different polypeptides and has a biconcave shape, and the size of hemoglobin is

13

about 65 Å; BSA consists of one polypeptide and has an ellipsoidal shape, and the

14

size of BSA was about 154 Å, larger than BHb.36 Therefore, BSA did not match with

15

the special recognition sites, and it could not effectively quench the fluorescence

16

intensity of the Si-NP/CdTe/MIP. Through the competitive experiments, it was further

17

confirmed that the Si-NP/CdTe/MIP had the specific recognition towards BHb.

18 19


20 21

3.4 Analysis and Detection of BHb in Real Samples 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

The practicability of Si-NP/CdTe/MIP was evaluated by selective separation and

2

enrichment of BHb from bovine blood and the SDS-PAGE was used to visualize

3

protein samples. As presented in Figure 8, the bands of BHb and BSA appeared in

4

300-fold dilution of bovine blood without treatment (lane 1). Lane 2 showed the

5

supernatant after treatment with Si-NP/CdTe/MIP, in which hardly any BHb was left.

6

Namely, almost all of the BHb was captured by Si-NP/CdTe/MIP. After elution with

7

Triton X-100, the band of BHb reappeared in lane 4, indicating BHb was enriched.

8

Compared with Si-NP/CdTe/MIP, the lane 3 and lane 5 showed the supernatant after

9

treatment with Si-NP/CdTe/NIP and the corresponding eluent with Triton X-100.

10

From lane 3, we can see about 75% BHb (quantification of the protein bands was

11

performed with Quantity One software (Bio-Rad)) still existed in the supernatant after

12

treatment with Si-NP/CdTe/NIP. The results further illustrated the specific recognition

13

sites were formed in Si-NP/CdTe/MIP and it has potential application for the selective

14

separation and enrichment of BHb in biological sample.

15 16


17 18

Specific and sensitive detection of proteins in practical and biological samples is

19

also one of the most important goals.37 To confirm the ability of the strategy to

20

sensitively detect target protein from real complex biological fluids, urine and bovine

21

blood were chosen and detected by the developed Si-NP/CdTe/MIP method. Firstly, 20


Page 20 of 38

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1

100-fold urine was used to detect and assess the analysis method and the accuracy of

2

the measurement system. As shown in Table 1, the recovery of 100-fold urine samples

3

is 98.7%-102.0%. And then, we spiked different concentration of BHb into 6000-fold

4

dilution of bovine blood, attaining recovery of 97.3%-104.0%. Overall, the result

5

showed the fluorescent sensor was successfully applied in the detection of BHb in

6

biological samples.

7 8

(Preferred position for Table 1)

9 10

4. Conclusions

11

In this work, a novel strategy was explored for the preparation of the

12

Si-NP/CdTe/MIP which possessed CdTe QDs as the fluorescence species and silica

13

nanoparticles as the support substrate for surface imprinting. The fluorescent

14

Si-NP/CdTe/MIP integrated the high sensitivity of QDs and the high selectivity of

15

MIT as well as the well-defined morphology, and was successfully used for the

16

selective recognition and determination of the template BHb in the biological samples.

17

The approach for synthesizing Si-NP/CdTe/MIP sensor provided an alternative

18

solution for special recognition and determination of protein in biological fluids as

19

well as might applied in more fields involving bio-diagnostics, biomedical and

20

proteomics research.

21 21



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1

Page 22 of 38

ACKNOWLEDGMENTS

2

This work was supported by the National Basic Research Program of China (973

3

Program) (Nos. 2011CB707703 and 2012CB910601), the National Natural Science

4

Foundation of China (No. 21075069 and 21275078), and the Tianjin Natural Science

5

Foundation (No. 11JCZDJC21700).

6 7 8 9 10

Electronic

Supplementary

Information

(ESI)

available:

[details

of

any

supplementary information available should be included here]. This information is available free of charge via the Internet at http://pubs.acs.org/.

11 12 13 14 15 16 17 18 19 20 21 22


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1

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Shea, K. J. J. Am. Chem. Soc. 2010, 132, 6644-6645.

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2007, 129, 7859-7866. (10) Verheyen, E.; Schillemans, J. P.; van Wijk, M.; Demeniex, M. A.; Hennink, W. E.; van Nostrum, C. F. Biomaterials 2011, 32, 3008-3020. (11)Zhang, W.; Qin, L.; He, X. W.; Li, W. Y.; Zhang, Y. K. J. Chromatogr. A 2009, 1216, 4560-4567. (12) Xia, Z.; Lin, Z.; Xiao, Y.; Wang, L.; Zheng, J.; Yang, H.; Chen, G. Biosens. Bioelectron. 2013, 47C, 120-126. 23



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Appl. Mater. Interfaces 2013, 5, 5208-5213. (16) Li, Z.; Barnes, J. C.; Bosoy, A.; Stoddart, J. F.; Zink, J. I. Chem. Soc. Rev. 2012, 41, 2590-2605. (17) Valizadeh, A.; Mikaeili, H.; Samiei, M.; Farkhani, S. M.; Zarghami, N.; Kouhi, M.; Akbarzadeh, A.; Davaran, S. Nanoscale Res. Lett. 2012, 7, 480. (18)Yu, K.; Zaman, B.; Romanova, S.; Wang, D. S.; Ripmeester, J. A. Small 2005, 1, 332-338.

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1

Figure captions

2

Scheme 1. Illustration for the preparation of the Si-NP/CdTe/MIP and the

3

fluorescence quenching detection of BHb upon specific recognition.

4

Figure 1. (a) FESEM image of Si-NP, (b) FESEM and (c) HRTEM images of

5

Si-NP/CdTe (Inset: high magnification HRTEM image of Si-NP/CdTe), and (d)

6

HRTEM image of Si-NP/CdTe/MIP.

7

Figure 2. The adsorption capacity of Si-NP/CdTe/MIP and Si-NP/CdTe/NIP.

8

Figure 3. The adsorption-desorption ability of Si-NP/CdTe/MIP and Si-NP/CdTe/NIP.

9

Figure 4. The pH-dependent FL intensity changes of Si-NP/CdTe/MIP and

10

Si-NP/CdTe/NIP by template BHb.

11

Figure 5. The fluorescence intensity change of Si-NP/CdTe/MIP within 60 min. Inset:

12

fluorescence photographs of Si-NP/CdTe/MIP in PBS buffer solution under visible

13

light (A) and under UV light irradiation (B).

14

Figure 6. Fluorescence emission spectra of Si-NP/CdTe/MIP (a) and Si-NP/CdTe/NIP

15

(b) with addition of the indicated concentrations of BHb. The Stern-Volmer plots of

16

Si-NP/CdTe/MIP (c) and Si-NP/CdTe/NIP (d) with BHb, respectively.

17

Figure 7. (a) Binding behaviors of different proteins (1.25 µM) on the

18

Si-NP/CdTe/MIP and Si-NP/CdTe/NIP. (b) Effects of the special competitive protein

19

BSA on the binding capacity under the different concentration of BSA and the fixed

20

concentration of BHb (1.25 µM) on the Si-NP/CdTe/MIP.

21

Figure 8. SDS-PAGE analysis of bovine blood samples before and after treatment by 27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Si-NP/CdTe/MIP and Si-NP/CdTe/NIP. Lane 0, protein marker; lane 1, 300-fold

2

dilution of bovine blood before treatment; lane 2, the supernatant after treatment with

3

Si-NP/CdTe/MIP; lane 3, the supernatant after treatment with Si-NP/CdTe/NIP; lane 4,

4

the eluent from Si-NP/CdTe/MIP; lane 5, the eluent from Si-NP/CdTe/NIP.

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 28


Page 28 of 38

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1

Scheme 1.

2 3 4 5 6 7 8 9 10 11 12 13 14 15 29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Figure 1.

(a)

(b)

(c)

(d)

2 3 4 5 6 7 8 9 10 11 12 30


Page 30 of 38

Page 31 of 38

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1

Figure 2.

2 3 4 5 6 7 8 9 10 11 12 13 31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Figure 3.

2 3 4 5 6 7 8 9 10 11 12 13 32


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1

Figure 4.

2

3 4

Figure 5.

5 6 7 8 9 10 11 12 13 33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Figure 6.

2

3 4 5 34


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1

Figure 7.

2

3 4 5 35



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1

Page 36 of 38

Figure 8. MW 94.0 66.2

0

1

2

3

4

5 BSA

45.0 35.0 25.0

18.4

14.4 BHb

2 3 4 5 6 7 8 9 10 11 12 13 14 36


Page 37 of 38

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1

Table 1 Results for the determination of the BHb in 100-fold dilution of urine and

2

6000-fold dilution of bovine blood.

3 BHb concentration (10-7 M) 4 5

Sample

6 7 8 9

urine

10 11 12 13

Measureda

Spiked

ndc 5.1±0.1 10.1±0.2 14.8±0.3

0.0 5.0 10.0 15.0

0.0 1.9 ±0.1 5.0 7.1 ±0.3 10.0 11.9±0.6 15.0 16.5±0.5 a b mean ± std, n=3; mean ± std recovery %, n=3. c Not detected.

%b

102.0±2.0 100.5±4.0 98.7±2.0

bovine blood

14 15 16 17 18 19 20 21 22 23 37


104.0 ±2.0 100.0 ±6.0 97.3±3.3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

TOC graphic

3 4

38


Page 38 of 38

Molecularly Imprinted Polymer

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