Aptamer Recognition Induced Target-Bridged Strategy for Proteins

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An aptamer recognition induced target-bridged strategy for proteins detection based on magnetic chitosan and silver/chitosan nanoparticles using surface-enhanced Raman spectroscopy Jincan He, Gongke Li, and Yuling Hu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03049 • Publication Date (Web): 05 Oct 2015 Downloaded from http://pubs.acs.org on October 6, 2015

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Analytical Chemistry

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An aptamer recognition induced target-bridged strategy for proteins

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detection

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nanoparticles using surface-enhanced Raman spectroscopy

based

on

magnetic

chitosan

and

silver/chitosan

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Jincan He, Gongke Li*, Yuling Hu*

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School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China

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* Corresponding author: Gongke Li, Yuling Hu

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Tel. : +86-20-84110922

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Fax : +86-20-84115107

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E-mail : [email protected]

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[email protected]

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ACS Paragon Plus Environment


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ABSTRACT: Poor selectivity and biocompability remain problems in applying for surface-

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enhance Raman spectroscopy (SERS) for direct detection of proteins due to similar spectra of

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most proteins and overlapping Raman bands in complex mixtures. To solve these problems, an

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aptamer recognition induced target-bridged strategy based on magnetic chitosan (MCS) and

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silver/chitosan nanoparticles (Ag@CS NPs) using SERS was developed for detection of protein

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benefiting from specific affinity of aptamer and biocompatibility of chitosan (CS). In this process,

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one aptamer (or antibody) modified MCS worked as capture probes through the affinity binding

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site of protein. The other aptamer modified with Raman report molecules encapsulated Ag@CS

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NPs was used as SERS sensing probe based on the other binding site of protein. The sandwich

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complexes of aptamer (antibody) /protein/aptamer were separated easily with a magnet from

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biological samples, and the concentration of protein was indirectly reflected by the intensity

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variation of SERS signal of Raman report molecules. To explore the universality of the strategy,

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three different kinds of proteins including thrombin, platelet derived growth factor BB (PDGF

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BB) and immunoglobulin E (lgE) were investigated. The major advantages of this aptamer

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recognition induced target-bridged strategy are convenient operation with a magnet, stable signal

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expressing resulting from preventing loss of report molecules with the help of CS shell, and the

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avoidance of slow diffusion-limited kinetics problems occurring on a solid substrate. To

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demonstrate the feasibility of the proposed strategy, the method was applied to detection of

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PDGF BB in clinical samples. The limit of detection (LOD) of PDGF BB was estimated to be 3.2

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pg/mL. The results obtained from human serum of healthy persons and cancer patients using the

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proposed strategy showed good agreement with that of the ELISA method, but with wider linear

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range, more convenient operation and lower cost. The proposed strategy holds great potential in

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highly sensitive and selective analysis of target proteins in complex biological samples.

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KEYWORDS: Target-bridged strategy; Aptamer recognition; Chitosan; Protein detection;

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Platelet derived growth factor; Surface-enhanced Raman spectroscopy; Biological sample. 2


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INTRODUCTION

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Surface enhanced Raman spectroscopy (SERS) is a vibrational spectroscopic technique for

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nondestructive and ultrasensitive detection of molecules on or near the surface of specific

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substrates, and greatly extends the role of standard Raman spectroscopy.1 The Raman intensity of

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molecules absorbing at rough metal surfaces increases by 106-1014 mainly due to electromagnetic,

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chemical, or electronic enhancement.2

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sensitivity with potential of single molecule detection and abundant informative spectra

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characteristics, making it promising method for biological detection.3,

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attracted tremendous interest in area of biochemistry and life sciences such as immunoassay,

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cellular studies, and cancer diagnosis.5-7 However, in some cases, it exhibits limited selectivity

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towards direct detecting biomolecules with high molecular weights such as proteins probably due

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to similar SERS spectra of these biomolecules and overlapping Raman bands in some complex

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mixtures.8,

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specific biorecognition and sensitive signal expression for selective detection of proteins.

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SERS has several unique advantages, such as high

4

Recently, SERS has

Therefore, it is quite necessary to develop extrinsic SERS labeling probes with

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Aptamers are artificial single-stranded DNA or RNA oligonucleotides selected from a huge

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combinatorial library by a process known as systematic evolution of ligands by exponential

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enrichment (SELEX).10 Aptamers are able to fold into secondary or three-dimensional complexes

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upon interaction with other molecules, which provides preferential binding sites for molecular

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recognition of analytes.11 Numerous aptamers with high affinity and high specificity have been

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selected against certain targets including small molecules, proteins, nucleic acids and even entire

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cells.12 Compared to antibodies which are traditionally used as recognition elements for detection,

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aptamers have obvious superiority, such as minimal immunogenicity, convenience of synthesis,

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ease of chemical modification, high stability and flexibility of the molecular structure, making

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them alternative candidates for affinity component of SERS probes.13

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Recently, a few SERS probes based on aptamer recognition for specific proteins detection have

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been reported.3, 11, 13, 14 One of the most popular technologies were the detection of the sandwich 3



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aptamer complexes immobilized on a solid substrate. In these cases, capture aptamers are

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immobilized on a solid substrate, and then antigens and probe aptamer-conjugated metal

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nanoparticles are sequentially added. Sandwich aptamer complexes are formed on the solid

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substrate. Afterwards, nonspecific binding antigens and aptamer-conjugated metal nanoparticles

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are washed out with buffer solution followed by SERS measurement. Target antigen biomarkers

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are quantitatively assayed by monitoring the intensity change of characteristic SERS peak of a

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report molecule labeled on the surface of metal nanoparticles.13

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Although this aptamer-based strategy greatly improved the selectivity of SERS detection, it has

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several drawbacks such as requirement of long incubation time in each binding step, loss of

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biological activity resulting from exposure to air and poor reproducibility of SERS signal.15 To

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resolve these problems, Yoon et al.

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SERS-based magnetic aptasensor. They used aptamer conjugated-magnetic beads as capturing

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probes and separation tools instead of a solid substrate, which overcame problems of slow

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reaction between aptamer and thrombin. However, both Raman active molecules and aptamers

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were physically attached to the metallic nanoparticles, which may lead to the dissociation of

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Raman active molecules in sample solution and interference of other molecules absorbing on the

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surface of metal nanoparticles in the complex matrices. Moreover, the applicability of

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quantitative determination was not conducted in real samples. A common way to protect Raman

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report molecules from dissociation is to coat metal NPs with a thin shell such as polymers16, 17,

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transition-metal materials15,

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biocompatibility or requirement for tedious modification steps impedes these materials for further

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application.

18, 19

11

developed a method for detection of thrombin using a

, carbon20,

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, and mesoporous silica13,

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. However, poor

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Chitosan (CS), an N-deacetylated derivative of chitin, is a well-known biopolymer with

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positively charged physicochemical and biocompatible characteristics.23 Abundant amino groups

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of chitosan not only make it biocompatible with organism but also facilitate it to bio-functionalize

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with proteins or amino-modified aptamers24-27. In the present study, by introducing aptamer and 4


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chitosan to improve selectivity and biocompability, we proposed an aptamer recognition induced

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target-bridged strategy for protein assay using aptamer modified Ag@CS NPs and aptamer (or

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antibody) modified MCS. The strategy relies on the availability of a pair of aptamers that bind to

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different regions of the protein. To demonstrate the universality, we investigated three different

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binding modes of the strategy taking thrombin, PDGF BB and lgE as model proteins. To explore

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the feasibility of the proposed strategy, it was employed for detection of PDGF BB in human

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serum samples. The proposed strategy has potential of recognizing the targets in complex

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matrices, and is convenient for rapid analysis of clinical samples with good selectivity,

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biocompability and high sensitivity.

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■EXPERIMENTAL SECTION

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Materials and reagents. Silver nitrate (≥99.8%), ferric trichloride (≥99.0%), sodium acetate

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(≥99.0%)

and

ethylene

glycol

(≥99.7%)

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from Guangzhou Chemical Reagent Factory (Guangzhou, China). Glutaraldehyde (25%, v/v) was

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purchased from Fuchen Chemical reagent Factory (Tianjin, China). Tris (hydroxymethyl)

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aminomethane (Tris, (≥99.9%)), 4-aminothiophenol (≥98.0%), chitosan with deacetylation

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degree of 95% and viscosity of 100-200 mPas were obtained from Aladdin Reagent Corporation

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(Shanghai, China). Amino-modified protein-binding aptamers in HPLC-purified form were

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synthesized in Sangon Biotechnology Inc. (Shanghai, China). TBA15 and TBA29 are aptamers

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binding to thrombin. TBA37 and TBA35 are aptamers binding to lgE and PDGF BB. The

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sequences of four employed aptamers are given below:

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TBA15: 5’-NH2-(CH2)6-GGTTGGTGTGGTTGG-3’

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TBA29: 5’-NH2-(CH2)6-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3’

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TBA37: 5’-NH2-(CH2)6-GGGGCACGTTTATCCGTCCCTCCTAGTGGCGTGCCCC-3’

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TBA35: 5’-NH2-(CH2)6-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3’

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Control TBA35: 5’-NH2-(CH2)6-GTGCGTACGGCACATTGTGATTCACCATGATCC-TG-

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3’. 5


were

purchased


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Serum samples were provided by Sun Yat-sen University Cancer Center (Guangzhou, China).

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Human thrombin (≥2,000 NIH units/mg, MW 37.4 kDa) was purchased from Sigma (St. Louis,

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MO, USA). Recombinant human platelet derived growth factor B-chain (PDGF-BB, MW 24.3

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kDa) was bought from Pepro Tech Inc. (New Jersey, USA). Human lgE protein and goat anti

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human lgE antibody were obtained from Fitzgerald (Acton, MA, USA). The commercial human

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PDGF BB enzyme-linked immunosorbent assay (ELISA) Kit was purchased from Xinfan

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Biological Technology Co., Ltd. (Shanghai, China). All reagents were of analytical grade and

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used without further purification.

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Instrument. Transmission electron microscopy (TEM) images were obtained on a FEI Tecnai

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G2 instrument (FEI, Netherlands). Infrared and UV-vis spectra were conducted on a NICOLET

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AVATAR 330 Fourier transform infrared spectrometer (Nicolet, USA) and a CARY 300Conc

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UV spectrophotometer (Varian, American), respectively. Zeta potential was measured on a

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Zetasizer Nano ZS90 (Malvern Ltd., Worcs,UK). X-ray diffractometry (XRD) was performed on

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D-MAX 2200 VPC (RIGAKU, Japan). ELISA experiments were measured on an iMark 168-

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1135 microplate reader (Bio-Rad, USA). Raman spectra were performed on a battery-powered

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Raman spectrometer (model Inspector Raman, diode laser, excitation wavelength λex = 785 nm)

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with the wavenumber range of 200-2000 cm-1 (DeltaNu, USA).

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Preparation of Ag@CS NPs linked with aptamers. Figure 1A shows the synthesis process

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of aptamer-Ag@CS NPs. Firstly, the Cit-AgNPs were prepared according to the procedure

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described by Lee and Meisel28. After cooling down, 20 µL of 1×10-4 mol/L 4-ATP was added to

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5 mL of cit-AgNPs at 35 ℃ under continuous stirring. The stirring was continued for 15 min. For

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the encapsulation of 4-ATP, 5 mL different concentrations (0.5, 1.0, 2.0, 4.0 mg/mL) of CS in 1%

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(v/v) acetic acid was added dropwise to the reaction mixture. The reaction was allowed to

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proceed for 30 min. Then, 1 mL of 2.5% glutaraldehyde (v/v) was added dropwise to the mixture

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under continuous stirring in 1 min. After reaction for another 2 h, the mixture was separated by

6


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centrifugation and washed several times with deionized water. The product was slowly re-

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dispersed into 5 mL of 10 mmol/L Tris-HCl buffer at pH 7.4.

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For the attachment of aptamer, 500 µL of 6.5 µmol/L aptamer (TBA15, TBA29, TBA35 or

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TBA37) was mixed with 2 mL of as-synthesized Ag@CS NPs dispersion and set in a shaker for

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overnight at room temperature. After that, the mixture was centrifuged and washed twice.

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Followed by addition of 0.1% BSA in Tris-HCl buffer and stirred for 1 h to block nonspecific

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adsorption. The precipitate was washed twice and re-dispersed into 1 mL of Tris-HCl buffer. The

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as-prepared TBA-Ag@CS NPs were stored at 4°C before use.

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Preparation of aptamer (or antibody)-conjugated MCS. MCS was synthesized by the

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hydrothermal method29. Briefly, sodium acetate and CS were added into mixture solution of

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ferric trichloride and ethylene glycol followed by stirring for 30 min. The whole mixture was

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transferred into Teflon-lined stainless-steel autoclave and reacted at 200 °C for 12 h.

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Approximately 2.0 mg of MCS were washed three times with Tris-HCl buffer before they were

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mixed with 1.0 mL of 2.5% glutaraldehyde solution in Tris-HCl buffer. The mixture was set in a

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shaker for 2 h at room temperature. The MCS was collected with a magnetic bar and washed

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three times with Tris-HCl buffer. Then aptamer (TBA15, TBA29 or TBA35) or anti-lgE antibody

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was added to the MCS with a final volume of 1.0 mL. The mixture was kept overnight in a shaker

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at room temperature. After that, the mixture was centrifuged and washed twice. Followed by

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addition of 0.1% BSA in Tris-HCl buffer and stirred for 1 h to block nonspecific adsorption. The

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aptamer-functionalized MCS were collected by a magnetic bar and washed twice with 1.0 mL

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Tris-HCl. The final product was stored in 1 mL of Tris-HCl at 4°C (Figure 1B).

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Surface-enhanced Raman scattering detection. In the first step, various concentrations of

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thrombin, PDGF BB and lgE solution were prepared with Tris-HCl containing 0.1% BSA,

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respectively. Then 25 µL of protein solution was added to 25 µL of corresponding TBA-MCS or

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antibody-MCS, and incubated for 30 min at room temperature. The resultant MCS were further

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reacted with 25 µL TBA-Ag@CS NPs for 1 h, and the sandwich aptamer complexes formed were 7



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subsequently isolated by applying a magnet to the wall of the microtube. The complexes were

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washed three times with 200 µL of Tris-HCl buffer using a micropipette. In this way, non-reacted

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reagents in residual solution were effectively removed. Afterwards, the target aptamer complexes

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were collected on the silicon wafer with a magnetic bar for SERS assay (Figure 1C).

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Application to analysis of PDGF BB in human serum. To explore the applicability of the

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proposed strategy in protein analysis, 6 human serum samples of 3 healthy persons and 3 cancer

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patients were assayed. All of the Raman spectra reported here was collected for 10 exposure

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seconds in the range of 800-1500 cm-1. For comparison, the serum samples were measured by a

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commercial ELISA kit with microplate reader. The procedures exactly followed the protocols

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suggested by the commercial kit. The optical density at the wavelength of 450 nm was detected.

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Control experiments were carried out to test the selectivity of this strategy. The SERS signals

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for negative control samples were measured at six groups of replacing PDGF BB with BSA, HRP,

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lgG, thrombin, lgE and replacing report TBA35 with control TBA35.

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■ RESULTS AND DISCUSSION

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In this study, we have developed an aptamer recognition induced target-bridged strategy using

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MCS and Ag@CS NPs for sensitive and selective detection of protein. The strategy relies on the

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availability of a pair of aptamers that bind to different regions of the protein. Capture aptamers

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(or antibodies) were firstly conjugated with MCS and used for recognizing protein, and then

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sensing aptamers linking with 4-ATP-embeded Ag@CS NPs was added for the formation of

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sandwich complexes of aptamer (antibody)/protein/aptamer. Once sandwich complexes were

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formed, concentration of protein was indirectly reflected by Raman intensity of 4-ATP. The

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schematic diagram of the sandwich-type assay is shown in Figure 1.

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Preparation and characterization of sensing probes and capture probes. Taking advantages

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of biocompatibility and abundant amino groups for further modification of CS, we prepared

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aptamer modified Ag@CS NPs as sensing probes and aptamer (or antibodiy) modified MCS as

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capture probes. 8


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Preparation and characterization of aptamer modified-Ag@CS NPs. It is well known that silver

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has larger optical cross section and is more inexpensive in comparison with that of gold. However,

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silver is susceptible to corrosion and easily affected by ambient factors, thereby weakening

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plasmonic signals and limiting its applications. In order to avoid aggregation and improve the

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stability of Ag NPs in the exposure of harsh environment, as well as to avoid dissociation of

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Raman report molecule, CS was coated onto the surface of Ag NPs after 4-ATP attached to Ag

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NPs.

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Figure 2 shows the TEM images of Ag@CS NPs prepared with CS of different concentrations.

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The thickness of CS layer on the surface of Ag NPs increased gradually with the increase of CS

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concentration from 0.5 mg/mL to 4.0 mg/mL. As the CS concentration increased, UV/vis

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absorption bands slightly red shifted (SI, Figure S1A), which further demonstrated the increase

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of the thickness as the increase of the concentration of CS. The concentration of CS plays a role

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in SERS intensity of 4-ATP. The strongest enhancement was observed at 0.5 mg/mL of CS (SI,

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Figure S1B). Slight decrease of SERS intensity took place with the concentration changing from

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1.0 to 2.0 mg/mL and obvious decrease was observed as the concentration of CS increased to 4.0

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mg/mL. Considering that thicker CS coating on Ag NPs took advantage in protecting 4-ATP

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from diffusion, we choose 2.0 mg/mL of CS in the following experiment. To investigate the

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stability of Ag@CS NPs, SERS performance of Ag@CS NPs and naked Ag NPs were measured

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and compared in presence of NaCl salt solution of different concentrations (SI, Figure S2). It

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depicted that Ag@CS NPs demonstrated significantly improved SERS stability in ionic

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circumstances.

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The amino groups in CS provide abundant amino group sites for covalent linkage of

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biomolecules such as amino-modified aptamers and antibodies to Ag@CS NPs and MCS via

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glutaraldehyde. To demonstrate whether aptamer was linked with Ag@CS NPs, IR spectra and ζ-

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potentials value were measured (SI, Figure S3). On one hand, after modification of Ag@CS NPs

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with glutaraldehyde, the peaks 3411 cm-1 and 3455 cm-1 (v-NH2) became weak and wide (SI, 9



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Figure S3A). The fact that 1588 cm-1 and 1624 cm-1 (δ-NH2) disappeared while 1636 cm-1 (v-

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C=N) appeared could be the excellent proof of cross-linking. Comparing curve c, d, e, f with b,

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1636 cm-1 (v-C=N) becoming evident and stronger demonstrated that aptamer was successfully

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conjugated with Ag@CS NPs. On the other hand, the ζ-potential was also performed (SI, Figure

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S3B). The ζ-potential of Ag NPs was -30.4 mV mainly due to negative charged citrate ions on the

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surface of Ag NPs. After coating Ag NPs with CS, the ζ-potential changed positive. After

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modification with aptamer, the ζ-potential turned negative, which demonstrated that Ag@CS NPs

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were successfully modified with aptamer.

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Preparation and characterization of aptamer (or antibody) modified-MCS. The MCS was

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synthesized through a solvothermal method. CS was used in the reaction system serving as both a

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ligand and a surface modification agent. Figure 3A shows representative SEM and TEM images

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of MCS, respectively. It is observed that the diameter of the spherical MCS is about 200 nm.

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Figure 3B depicts that the saturation magnetization of the MCS was 35.5 emu /g, which was

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enough for magnetic separation with a magnet bar. The XRD pattern of MCS is shown in Figure

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3C, which can be indexed to Fe3O4. Figure 3D presents the FTIR spectra of CS and MCS. In

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addition to characteristic bands of CS observing at 3435 cm-1 (v-O-H and v-N-H), 1624 cm-1 (δ-

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N-H) and 1424 cm-1 (v-C-N), and 1089 cm-1 (v-C-O-C), a band of 577 cm-1 related to v-Fe-O

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appearing in MCS, which indicated that CS was modified successfully with Fe3O4.

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In order to modify MCS with aptamer (or antibody), MCS was firstly functionalized with

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glutaraldehyde. Residual aldehyde groups reacted with amino groups of aptamer or anti-lgE

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antibody. FTIR spectra of MCS, glutaraldehyde modified-MCS, TBA15-MCS, TBA29-MCS,

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TBA35-MCS and anti-lgE antibody-MCS were performed to demonstrate the modification

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process (SI, Figure S4). Compared curve a and b, after functionalization of MCS with

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glutaraldehyde, the peak 1625 cm-1 (δ-NH2) disappeared while 1705 cm-1(v-CHO) and 1635 cm-1

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(v-C=N) appeared, which was the excellent proof of modification of glutaraldehyde. We observed

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that the peaks 1705 cm-1(v-CHO) disappeared and 1635 cm-1 (v-C=N) became stronger in curve c, 10


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d, e and f, which indicated that aptamer and anti-lgE antibody were modified onto MCS

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successfully.

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Development of the aptamer recognition induced target-bridged strategy for protein

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detection. Figure 4 shows the overall steps for the formation of sandwich aptamer complexes

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based on the aptamer recognition induced target-bridged strategy. Firstly, aptamer modified MCS

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selectively recognized and captured proteins benefiting from biocompatibility of CS and affinity

259

of aptamer. After aptamer conjugated Ag@CS NPs were added, the sandwich complexes of

260

aptamer/protein/aptamer were formed based on aptamer–antigen–aptamer interactions. These

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complexes were isolated by a magnetic bar, washed, and re-dispersed in Tris-HCl buffer, the

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residual Ag@CS NPs in the solution failing to form aptamer complexes were eventually removed

263

by washing step. Strong SERS signals of 4-ATP were observed due to SERS effect of Ag NPs in

264

the exposure of laser (Figure 4A). However, in the absence of protein, it failed to form the

265

sandwich aptamer complexes, thus no SERS signal was observed (Figure 4B).

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In this approach, the analyte was sandwiched by a pair of aptamers, one capture aptamer and the

267

other report aptamer. Capture aptamers were immobilized on the surface of MCS, while report

268

aptamers were conjugated with 4-ATP embedded Ag@CS NPs as signaling moieties. In

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consideration of various combination styles between protein and binding aptamer, three different

270

kinds of protein including thrombin, PDGF BB and lgE were investigated. Figure 5 depicted

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three modes of the aptamer recognition induced target-bridged strategy.

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Detection of thrombin. In most cases, capture and report aptamers of proteins have different

273

nucleic acid sequences such as thrombin, which has one 15-mer DNA aptamer binding to the

274

fibrinogen-recognition exosite and the other 29-mer DNA aptamer binding to the heparin-binding

275

exosite. It is common to fabricate sandwich complex of TBA15 /thrombin/TBA29 for thrombin

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assay. In consideration of two different aptamer binding sites of thrombin, we compared different

277

binding modes of aptamers and NPs, that are TBA15-Ag@CS/thrombin/MCS-TBA29 and

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TBA29-Ag@CS/thrombin/MCS-TBA15. It was found that the SERS signal of TBA2911



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Ag@CS/thrombin/MCS-TBA15 is stronger than that of TBA15-Ag@CS/thrombin/MCS-TBA29

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(SI, Figure S5). For the purpose of high sensitivity, therefore, it is preferable to modify Ag@CS

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NPs with one aptamer of higher affinity and modify MCS with the other.

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Detection of PDGF BB. Specifically, in limited cases, some proteins such as PDGF BB are

283

dimeric molecule and contain two identical aptamer binding sites, thus allowing the use of a

284

single aptamer for the sandwich formation. Taking PDGF BB as an example, TBA35 was firstly

285

immobilized onto Ag@CS NPs and MCS respectively. Excess TBA35-MCS bound with PDGF

286

BB through one binding site and selectively captured PDGF BB from solution through protein-

287

aptamer interaction. When TBA35-Ag NPs were added, it linked with PDGF BB through the

288

other binding site, thus the sandwich complex of aptamer/PDGF BB/aptamer was formed.

289

Detection of immunoglobulin E. Also of note, in cases when there are no two aptamers sharing

290

identical or overlapping binding sites on protein such as lgE, it is possible to use an antibody as

291

the second ‘aptamer’ to form sandwich complex of aptamer/protein/antibody. Human IgE has

292

two different binding sites of TBA37 and antibody. For the assay, goat anti-human IgE antibody

293

was immobilized onto the surface of MCS and TBA37 was modified onto Ag@CS NPs

294

respectively through the link of glutaraldehyde. After human IgE was added, human IgE bound

295

with its antibody by the antigen-antibody interaction through one binding site. TBA37-Ag NPs

296

were attached to lgE to form sandwich of aptamer/lgE/antibody through the other binding site.

297

With the proposed approach, different proteins can be selectively measured through changing

298

binding aptamers (or antibody) on the surface of Ag@CS NPs and MCS. Reproducible SERS

299

spectra of 4-ATP with strong signal were obtained from detection of different proteins in Figure

300

5. Both of them show characteristic of ~1077 cm-1, ~1176 cm-1 , and ~1576 cm-1, which are

301

assigned to stretching vibration of C-S, bending vibration of C-S and stretching vibration of C-C,

302

respectively. Therefore, the labeled approach for protein detection overcomes the problem of

303

poor selectivity and sensitivity occurring in label-free method. Moreover, it solves problem of

304

diffusion-limited kinetics on solid substrate and can be conveniently operated with a magnet bar. 12


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305

Application for PDGF BB detection in human serum. To explore the feasibility of the

306

proposed strategy, the detection of PDGF BB in human serum sample was performed. PDGF BB

307

is a well-characterized growth factor displaying potent biological effects on angiogenesis30.

308

Recent studies reveal that overexpression of PDGF BB within tumors results in increased

309

pericyte coverage, suggesting that PDGF BB signaling is also essential for tumor growth and

310

cancerous pericyte recruitment process31. Therefore, the detection of PDGF BB in human serum

311

is significant for early diagnosis and treatment of cancer.

312

Reproducibility. SERS spectra of the same aptamer/PDGF BB/aptamer complexes at seven

313

different spots were collected and the intensity at 1077 cm-1 was used to test the reproducibility.

314

The coefficient of variation was less than 8% (Figure 6A). It shows that the sandwich complexes

315

possess good reproducibility. It is very likely that, on one hand, the CS shell showed excellent

316

biocompatibility and protected Raman report molecules from dissociation in harsh environment.

317

On the other hand, the formed sandwich complexes of aptamer/PDGF BB/aptamer were

318

homogeneous (Figure 6B) so that reproducible signal was obtained at different spots.

319

Sensitivity and Selectivity. Figure 6C shows SERS spectra of 4-ATP in the presence of various

320

concentrations of PDGF BB and their corresponding calibration curves. The SERS signal of 4-

321

ATP at 1077 cm-1 increased upon the increasing concentration of PDGF BB ranging from 10

322

pg/mL to 5.0 ng/mL (0.4-2.0×102 pmol/L). The relative Raman intensity of 4-ATP at 1077 cm-1

323

was monitored and used as a quantitative evaluation of the PDGF BB antigen levels. Figure 6C

324

(inset) shows good linear response between intensity of 4-ATP and concentration of PDGF BB

325

with correlation coefficient of 0.9937. The limit of detection (LOD) was estimated to be 3.2

326

pg/mL (0.1 pmol/L). In order to test the selectivity of this strategy, six groups of contrast

327

experiments were carried out through replacing PDGF BB with BSA, HRP, IgG, thrombin, IgE

328

and replacing report TBA35 on Ag NPs with control TBA35, respectively (SI, Figure S6). It

329

showed that negligible Raman signal was obtained by replacing the proteins with BSA, HRP and

330

IgG, thrombin or lgE due to the failure formation of sandwich complex. Moreover, no obvious 13



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

331

signal was obtained replacing the report TBA35 on Ag@CS NPs with control TBA35. Strong

332

signal was obtained only when the specific protein PDGF BB and corresponding aptamer existed.

333

The assay was very selective to PDGF BB over the tested interferences, mainly due to

334

simultaneous recognition and high specificity between a pair of affinity aptamer probes and

335

PDGF BB.

336

Detection of PDGF BB in human serum. To test the applicability of this assay, 6 human serum

337

samples collected from 3 healthy persons and 3 cancer patients were assayed by the proposed

338

method using ELISA as the reference method. Human serum samples have been diluted 5 times

339

with Tris-HCl buffer. It showed that concentrations of PDGF BB in serum of cancer patients

340

were obviously higher than that in healthy persons (Table 1). In addition, the results obtained

341

with the proposed strategy were in good agreement with those obtained by ELISA but with wider

342

linear range, more convenient operation and lower cost. Moreover, it compares favorably with

343

those of most optical approaches14, 32-39 for detection of PDGF BB (Table 2). Remarkably, the

344

proposed aptamer recognition induced target-bridged strategy for PDGF BB detection is more

345

sensitive and has wider linear range than the previous SERS-based method14 probably due to

346

biocompatibility of chitosan and protection of Raman report molecules from loss with the help of

347

chitosan.

348 349

■ CONCLUSION

350

In the present study, we have developed an aptamer recognition induced target-bridged strategy

351

based on Ag@CS NPs and MCS for detection of proteins. The chitosan shell and specific affinity

352

of aptamers provide guarantees of selectivity and sensitivity of the strategy. To explore the

353

universality of the strategy, three different kinds of proteins including thrombin, PDGF BB and

354

lgE as three model proteins were investigated. The strategy is suitable for detection of three kinds

355

of proteins with two different aptamer binding sites or two identical binding sites or one aptamer

356

binding site and another antibody binding site. To demonstrate the feasibility of the strategy, it 14


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357

was employed for detection of PDGF BB in human serum samples. The present SERS approach

358

provides advantages of high sensitivity and accuracy towards PDGF BB detection, which mainly

359

attributes to biocompatibility of CS and specific recognition of aptamer. The LOD for PDGF BB

360

was as low as 3.2 pg/mL and the determined results were consistent with that of the ELISA

361

method, but with wider linear range, convenient operation and low cost. As more aptamer being

362

selected for proteins, we envision that the strategy would be a universal, convenient, selective and

363

sensitive clinical tool for detection of proteins in complicated biological samples.

364 365

■ ASSOCIATED CONTENT

366

Supporting Information

367

Additional information as noted in text. This material is available free of charge via the Internet

368

at http://pubs.acs.org

369

■ AUTHOR INFORMATION

370

Corresponding Author

371

*E-mail: [email protected]

372

*E-mail: [email protected]

373

Notes

374

The authors declare no competing financial interest.

375 376 377

■ ACKNOWLEDGMENTS The authors would like to thank Major National Scientific Instrument and Equipment

378

Development Project (2011YQ03012409), and the National Natural Science Foundation of China

379

for financially supporting this research under grant numbers 21277176, 21475153 and 21127008,

380

respectively. 15



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REFERENCES (1) Hu, Y. L.; Liao, J.; Wang, D. M.; Li, G. K. Anal. Chem. 2014, 86, 3955-3963.

(2) Deng, Z.; Chen, X. X.; Wang, Y. R.; Fang, E. H.; Zhang, Z. G.; Chen, X. Anal. Chem. 2015, 87, 633-

640.

(3) Zheng, J.; Hu, Y. P.; Bai, J. H.; Ma, C.; Li, J. S.; Li, Y. H.; Shi, M. L.; Tan, W. H.; Yang, R. H. Anal.

Chem. 2014, 86, 2205-2212.

(4) Zhang, K. G.; Yao, S.; Li, G. K.; Hu, Y. L. Nanoscale 2015, 7, 2659-2666.

(5) Wang, Y. L.; Vaidyanathan, R.; Shiddiky, M. J. A.; Trau, M. ACS Nano 2015, 9, 6354-6362.

(6) Narayanan, N.; Karunakaran, V.; Paul, W.; Venugopal, K.; Sujathan, K.; Kumar Maiti, K. Biosens.

Bioelectron. 2015, 70, 145-152.

(7) Karthigeyan, D.; Siddhanta, S.; Kishore, A. H.; Perumal, S. S. R. R.; Agren, H.; Sudevan, S.; Bhat, A.

V.; Balasubramanyam, K.; Subbegowda, R. K.; Kundu, T. K.; Narayana, C. P. Natl. Acad. Sci. USA 2014,

111, 10416-10421.

(8) Han, X. X.; Chen, L.; Guo, J.; Zhao, B.; Ozaki, Y. Anal. Chem. 2010, 82, 4102-4106.

(9) Balzerova, A.; Fargasova, A.; Markova, Z.; Ranc, V.; Zboril, R. Anal. Chem. 2014, 86, 11107-11114.

(10) Du, F. Y.; Guo, L.; Qin, Q.; Zheng, X.; Ruan, G. H.; Li, J. P.; Li, G. K. Trends Anal. Chem. 2015, 67,

134-146.

(11) Yoon, J.; Choi, N.; Ko, J.; Kim, K.; Lee, S.; Choo, J. Biosens. Bioelectron. 2013, 47, 62-67.

(12) Li, Y.; Qi, X. D.; Lei, C. C.; Yue, Q. F.; Zhang, S. S. Chem. Commun. 2014, 50, 9907-9909.

(13) Chen, X.; Liu, H. L.; Zhou, X. D.; Hu, J. M. Nanoscale 2010, 2, 2841-2846. 16


Page 16 of 29

Page 17 of 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


(14) Wang, C. W.; Chang, H. T. Anal. Chem. 2014, 86, 7606-7611.

(15) Chon, H.; Lee, S.; Son, S. W.; Oh, C. H.; Choo, J. Anal. Chem. 2009, 81, 3029-3034.

(16) Yang, M. X.; Chen, T.; Lau, W. S.; Wang, Y.; Tang, Q. H.; Yang, Y. H.; Chen, H. Y. Small 2009, 5,

198-202.

(17) Shkilnyy, A.; Soucé, M.; Dubois, P.; Warmont, F.; Saboungi, M.; Chourpa, I. Analyst 2009, 134, 1868-

1872.

(18) Kang, H.; Jeong, S.; Park, Y.; Yim, J.; Jun, B. H.; Kyeong, S.; Yang, J. K.; Kim, G.; Hong, S. G.; Lee,

L. P.; Kim, J. H.; Lee, H. Y.; Jeong, D. H.; Lee, Y. S. Adv. Funct. Mater. 2013, 23, 3719-3727.

(19) Ma, W.; Yin, H. H.; Xu, L. G.; Wu, X. L.; Kuang, H.; Wang, L. B.; Xu, C. L. Chem. Commun. 2014, 50,

9737-9740.

(20) Shen, A. G.; Chen, L. F.; Xie, W.; Hu, J. C.; Zeng, A.; Richards, R.; Hu, J. M. Adv. Funct. Mater. 2010,

20, 969-975.

(21) Liu, F. X.; Cao, Z. S.; Tang, C. J.; Chen, L.; Wang, Z. L. ACS Nano 2010, 4, 2643-2648.

(22) Zheng, C.; Liang, L. J.; Xu, S. P.; Zhang, H. P.; Hu, C. X.; Bi, L. R.; Fan, Z. M.; Han, B.; Xu, W. Q.

Anal. Bioanal. Chem. 2014, 406, 5425-5432.

(23) He, J. C.; Zhou, F. Q.; Mao, Y. F.; Tang, Z. N.; Li, C. Y. Anal. Lett. 2013, 46, 1430-1441.

(24) Jeong, S.; Choi, S. Y.; Park, J.; Seo, J.; Park, J.; Cho, K.; Joo, S.; Lee, S. Y. J. Mater. Chem. 2011, 21,

13853-13859.

(25) Han, X. X.; Schmidt, A. M.; Marten, G.; Fischer, A.; Weidinger, I. M.; Hildebrandt, P. ACS Nano 2013,

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

7, 3212-3220.

(26) Potara, M.; Gabudean, A.; Astilean, S. J. Mater. Chem. 2011, 21, 3625-3633.

(27) Potara, M.; Boca, S.; Licarete, E.; Damert, A.; Alupei, M.; Chiriac, M. T.; Popescu, O.; Schmidt, U.;

Astilean, S. Nanoscale 2013, 5, 6013-6022.

(28) Lee, P. C.; Meisel, D. J. Phys. Chem. 1982, 86, 3391-3395.

(29) Gao, L. Z.; Wu, J. M.; Lyle, S.; Zehr, K.; Cao, L. L.; Gao, D. J. Phys. Chem. C 2008, 112, 17357-17361.

(30)

Lange, S.; Heger, J.; Euler, G.; Wartenberg, M.; Piper, H. M.; Sauer, H. Cardiovasc. Res. 2008, 81,

159-168.

(31)

Song, N.; Huang, Y. J.; Shi, H. B.; Yuan, S. P.; Ding, Y. P.; Song, X. M.; Fu, Y.; Luo, Y. Z.

Cancer Res. 2009, 69, 6057-6064.

(32) Chang, C. C.; Wei, S. C.; Wu, T. H.; Lee, C. H.; Lin, C. W. Biosens. Bioelectron. 2013, 42, 119-123.

(33) Li, H.; Zhu, Y.; Dong, S. Y.; Qiang, W. B.; Sun, L.; Xu, D. K. Anal. Chim. Acta 2014, 829, 48-53.

(34) Liu, J. J.; Song, X. R.; Wang, Y. W.; Zheng, A. X.; Chen, G. N.; Yang, H. H. Anal. Chim. Acta 2012,

749, 70-74.

(35) Liang, J. F.; Wei, R.; He, S.; Liu, Y. K.; Guo, L.; Li, L. D. Analyst 2013, 138, 1726-1732.

(36) Li, H.; Wang, M.; Wang, C. Z.; Li, W.; Qiang, W. B.; Xu, D. K. Anal. Chem. 2013, 85, 4492-4499.

(37) Wang, P.; Song, Y. H.; Zhao, Y. J.; Fan, A. P. Talanta 2013, 103, 392-397.

(38) Bi, S.; Luo, B. Y.; Ye, J. Y.; Wang, Z. H. Biosens. Bioelectron. 2014, 62, 208-213.

(39) Zhang, X. F.; Zhang, H.; Xu, S. X.; Sun, Y. H. Analyst 2013, 139, 133-137.

18


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Figure captions Figure 1 Schematic representation of aptamer recognition induced target-bridged strategy for protein detection using aptamer modified-MCS as capture probes and aptamer conjugated 4-ATP embedded Ag@CS NPs as sensing probes.

Figure 2 TEM images of 4-ATP embedded Ag@CS NPs prepared with CS of different concentrations: (A) 0.5 mg/mL, (B) 1.0 mg/mL, (C) 2.0 mg/mL, (D) 4.0 mg/mL.

Figure 3 (A) TEM image and SEM image of MCS. (B) Magnetization curve of MCS. (C) XRD pattern of MCS. (D) FTIR spectra of CS and MCS.

Figure 4 Schematic illustration of the whole procedure of the aptamer recognition induced target-bridged strategy : (A) with protein and (B) without protein.

Figure 5 Three modes of aptamer recognition induced target-bridged strategy for respective detection of thrombin, PDGF BB and lgE.

Figure 6 (A) SERS intensity measured from complexes of aptamer/PDGF BB/aptamer at 1077 cm −1 at seven different spots (CPDGF BB =100 pg/mL). (B) TEM of sandwich complexes of aptamer/PDGF BB/aptamer (CPDGF BB =100 pg/mL). (C) SERS spectra obtained in presence of PDGF BB with different concentrations (the concentrations from bottom to top (a−g) are blank, 10.0 pg/mL, 50.0 pg/mL, 0.1 ng/mL, 0.5 ng/mL, 1.0 ng/mL, and 5.0 ng/mL). Inset: the calibration curve for the SERS intensity at 1077 cm−1 as a function of the logarithmic concentration of PDGF BB (R=0.9937). (D) SERS detection of PDGF BB in blank solution (a), serum sample (b) and serum sample spiked at 750 pg/mL (c). 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

Figure 1 Schematic representation of aptamer recognition induced target-bridged strategy for protein detection using aptamer modified-MCS as capture probes and aptamer conjugated 4-ATP embedded Ag@CS NPs as sensing probes.

20


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Figure 2 TEM images of 4-ATP embedded Ag@CS NPs prepared with CS of different concentrations: (A) 0.5 mg/mL, (B) 1.0 mg/mL, (C) 2.0 mg/mL, (D) 4.0 mg/mL.

21



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

Figure 3 (A) TEM image and SEM image of MCS. (B) Magnetization curve of MCS. (C) XRD pattern of MCS. (D) FTIR spectra of CS and MCS.

22


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Figure 4 Schematic illustration of the whole procedure of the aptamer recognition induced target-bridged strategy: (A) with protein and (B) without protein.

23



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

Figure 5 Three modes of aptamer recognition induced target-bridged strategy for respective detection of thrombin, PDGF BB and lgE.

24


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Figure 6 (A) SERS intensity measured from complexes of aptamer/PDGF BB/aptamer at 1077 cm −1 at seven different spots (CPDGF BB =100 pg/mL). (B) TEM of sandwich complexes of aptamer/PDGF BB/aptamer (CPDGF BB =100 pg/mL). (C) SERS spectra obtained in presence of PDGF BB with different concentrations (the concentrations from bottom to top (a−g) are blank, 10.0 pg/mL, 50.0 pg/mL, 0.1 ng/mL, 0.5 ng/mL, 1.0 ng/mL, 5.0 ng/mL). Inset: the calibration curve for the SERS intensity at 1077 cm−1 as a function of the logarithmic concentration of PDGF BB (R=0.9937). (D) SERS detection of PDGF BB in blank solution (a), serum sample (b) and serum sample spiked at 750 pg/mL (c).

25



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Table captions Table 1 PDGF BB concentration (pg/mL) in human serum samples detected by the proposed SERS method and ELISA method.

Table 2 Comparison of different optical aptasensors for PDGF-BB determination.

26


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Table 1 PDGF-BB concentration (pg/mL) in human serum samples detected by the proposed SERS method and ELISA method.

SERS Samplea

ELISA

Determinedb

Added

Recovery

Determinedc

(pg/mL)

(pg/mL)

(%)

(pg/mL)

150.0

117.0

750.0

99.5

150.0

88.6

750.0

93.1

150.0

119.3

750.0

89.7

1000

87.5

3000

76.8

1000

90.7

3000

84.4

1000

102.2

3000

77.3

Relative error (%)

Serum1

Serum 2

Serum 3

Serum 4

Serum 5

Serum 6

a

142.6±7.1

101.7±1.9

177.8±7.3

921.6±68.2

836.2±52.7

782.4±64.2

156.6±6.2

-8.9

109.8±4.8

-7.4%

169.2±8.5

5.1

872.4±62.6

5.6%

915.3±48.2

-8.6%

726.3±58.1

7.7%

Samples of 1 to 3 were from healthy persons and samples of 4 to 6 were from cancer

patients. b

Quantitative data was integrated at 1077 cm-1. The data were from three independent

experiments. c

The data were collected from three independent experiments.

27



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Page 28 of 29

Table 2 Comparison of different optical aptasensors for PDGF-BB determination.

methods

colorimetric

fluorescence

chemiluminescent

probes

LOD

Linearity range

(pmol/L)

(nmol/L)

Apt and Au NPs

6000

10-1000

32

Apt and Ag NPs

200

0.2-8.2

33

Apt and Ag NCs

370

1-50

34

FAM-Apt, GO

167

0.167-1.167

35

Apt-Ag NPs

27

0.2-17

36

Apt, Au NPs and luminol

60

0.06-6

37

0.68

1×10-3-10

38

FITC, luminol

50

0.1-100

39

Au PNNs, Apt/MBA-Au NPs

0.5

1×10-3-5×10-2

14

TBA-Ag@CS NPs and TBA-MCS

0.1a

4×10-4 -0.2a

Apt, hemin, luminal and DNAzyme

SERS

ref

This method

a

The mole concentration was obtained from dividing mass concentration by molecular

weight of PDGF BB.

28


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For TOC only:

29


Aptamer Recognition Induced Target-Bridged Strategy for Proteins

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