Aptamer Recognition Induced Target-Bridged Strategy for Proteins

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

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S Supporting Information *

ABSTRACT: Poor selectivity and biocompability remain problems in applying surface-enhanced Raman spectroscopy (SERS) for direct detection of proteins due to similar spectra of most proteins and overlapping Raman bands in complex mixtures. To solve these problems, an aptamer recognition induced target-bridged strategy based on magnetic chitosan (MCS) and silver/chitosan nanoparticles (Ag@CS NPs) using SERS was developed for detection of protein benefiting from specific affinity of aptamers and biocompatibility of chitosan (CS). In this process, one aptamer (or antibody) modified MCS worked as capture probes through the affinity binding site of protein. The other aptamer modified Raman report molecules encapsulated Ag@CS NPs were used as SERS sensing probes based on the other binding site of protein. The sandwich complexes of aptamer (antibody)/protein/aptamer were separated easily with a magnet from biological samples, and the concentration of protein was indirectly reflected by the intensity variation of SERS signal of Raman report molecules. To explore the universality of the strategy, three different kinds of proteins including thrombin, platelet derived growth factor BB (PDGF BB) and immunoglobulin E (lgE) were investigated. The major advantages of this aptamer recognition induced target-bridged strategy are convenient operation with a magnet, stable signal expressing resulting from preventing loss of report molecules with the help of CS shell, and the avoidance of slow diffusionlimited kinetics problems occurring on a solid substrate. To demonstrate the feasibility of the proposed strategy, the method was applied to detection of PDGF BB in clinical samples. The limit of detection (LOD) of PDGF BB was estimated to be 3.2 pg/mL. The results obtained from human serum of healthy persons and cancer patients using the proposed strategy showed good agreement with that of the ELISA method but with wider linear range, more convenient operation, and lower cost. The proposed strategy holds great potential in highly sensitive and selective analysis of target proteins in complex biological samples.

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Aptamers are artificial single-stranded DNA or RNA oligonucleotides selected from a huge combinatorial library by a process known as systematic evolution of ligands by exponential enrichment (SELEX).10 Aptamers are able to fold into secondary or three-dimensional complexes upon interaction with other molecules, which provides preferential binding sites for molecular recognition of analytes.11 Numerous aptamers with high affinity and high specificity have been selected against certain targets including small molecules, proteins, nucleic acids, and even entire cells.12 Compared to antibodies which are traditionally used as recognition elements for detection, aptamers have obvious superiority, such as minimal immunogenicity, convenience of synthesis, ease of chemical modification, high stability, and flexibility of the molecular structure, making them alternative candidates for the affinity component of SERS probes.13

urface enhanced Raman spectroscopy (SERS) is a vibrational spectroscopic technique for nondestructive and ultrasensitive detection of molecules on or near the surface of specific substrates, and greatly extends the role of standard Raman spectroscopy.1 The Raman intensity of molecules absorbing at rough metal surfaces increases by 106−1014 mainly due to electromagnetic, chemical, or electronic enhancement.2 SERS has several unique advantages, such as high sensitivity with the potential of single molecule detection and abundant informative spectra characteristics, making it a promising method for biological detection.3,4 Recently, SERS has attracted tremendous interest in area of biochemistry and life sciences such as immunoassay, cellular studies, and cancer diagnosis.5−7 However, in some cases, it exhibits limited selectivity toward direct detecting biomolecules with high molecular weights such as proteins probably due to similar SERS spectra of these biomolecules and overlapping Raman bands in some complex mixtures.8,9 Therefore, it is quite necessary to develop extrinsic SERS labeling probes with specific biorecognition and sensitive signal expression for selective detection of proteins. © 2015 American Chemical Society

Received: August 8, 2015 Accepted: October 5, 2015 Published: October 5, 2015 11039

DOI: 10.1021/acs.analchem.5b03049 Anal. Chem. 2015, 87, 11039−11047

Article

Analytical Chemistry

(Tianjin, China). Tris(hydroxymethyl) aminomethane (Tris, (≥99.9%)), 4-aminothiophenol (≥98.0%), chitosan with deacetylation degree of 95%, and viscosity of 100−200 mPa s were obtained from Aladdin Reagent Corporation (Shanghai, China). Amino-modified protein-binding aptamers in HPLCpurified form were synthesized in Sangon Biotechnology Inc. (Shanghai, China). TBA15 and TBA29 are aptamers binding to thrombin. TBA37 and TBA35 are aptamers binding to lgE and PDGF BB, respectively. The sequences of four employed aptamers are given below: TBA15: 5′-NH2-(CH2)6-GGTTGGTGTGGTTGG-3′ TBA29: 5′-NH2-(CH2)6-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3′ TBA37: 5′-NH 2 -(CH 2 ) 6 -GGGGCACGTTTATCCGTCCCTCCTAGTGGCGTGCCCC-3′ TBA35: 5′-NH2-(CH2)6-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3′ Control TBA35: 5′-NH2-(CH2)6-GTGCGTACGGCACATTGTGATTCACCATGATCC-TG-3′. Serum samples were provided by Sun Yat-sen University Cancer Center (Guangzhou, China). Human thrombin (≥2 000 NIH units/mg, MW 37.4 kDa) was purchased from Sigma (St. Louis, MO). Recombinant human platelet derived growth factor B-chain (PDGF-BB, MW 24.3 kDa) was bought from Pepro Tech Inc. (New Jersey). Human lgE protein and goat anti human lgE antibody were obtained from Fitzgerald (Acton, MA). The commercial human PDGF BB enzymelinked immunosorbent assay (ELISA) Kit was purchased from Xinfan Biological Technology Co., Ltd. (Shanghai, China). All reagents were of analytical grade and used without further purification. Instrument. Transmission electron microscopy (TEM) images were obtained on a FEI Tecnai G2 instrument (FEI, The Netherlands). Infrared and UV−vis spectra were conducted on a NICOLET AVATAR 330 Fourier transforminfrared spectrometer (Nicolet) and a CARY 300Conc UV spectrophotometer (Varian), respectively. The zeta potential was measured on a Zetasizer Nano ZS90 (Malvern Ltd., Worcs, U.K.). X-ray diffractometry (XRD) was performed on D-MAX 2200 VPC (RIGAKU, Japan). ELISA experiments were measured on an iMark 168-1135 microplate reader (BioRad). Raman spectra were performed on a battery-powered Raman spectrometer (model Inspector Raman, diode laser, excitation wavelength λex = 785 nm) with the wavenumber range of 200−2000 cm−1 (DeltaNu). Preparation of Ag@CS NPs Linked with Aptamers. Figure 1A shows the synthesis process of aptamer-Ag@CS NPs. First, the Cit-AgNPs were prepared according to the procedure described by Lee and Meisel.28 After cooling down, 20 μL of 1 × 10−4 mol/L 4-ATP was added to 5 mL of cit-AgNPs at 35 °C under continuous stirring. The stirring was continued for 15 min. For the encapsulation of 4-ATP, 5 mL of different concentrations (0.5, 1.0, 2.0, 4.0 mg/mL) of CS in 1% (v/v) acetic acid was added dropwise to the reaction mixture. The reaction was allowed to proceed for 30 min. Then, 1 mL of 2.5% glutaraldehyde (v/v) was added dropwise to the mixture under continuous stirring in 1 min. After reaction for another 2 h, the mixture was separated by centrifugation and washed several times with deionized water. The product was slowly redispersed into 5 mL of 10 mmol/L Tris-HCl buffer at pH 7.4. For the attachment of aptamer, 500 μL of 6.5 μmol/L aptamer (TBA15, TBA29, TBA35, or TBA37) was mixed with 2 mL of as-synthesized Ag@CS NPs dispersion and set in a

Recently, a few SERS probes based on aptamer recognition for specific proteins detection have been reported.3,11,13,14 One of the most popular technologies were the detection of the sandwich aptamer complexes immobilized on a solid substrate. In these cases, capture aptamers are immobilized on a solid substrate, and then antigens and probe aptamer-conjugated metal nanoparticles are sequentially added. Sandwich aptamer complexes are formed on the solid substrate. Afterward, nonspecific binding antigens and aptamer-conjugated metal nanoparticles are washed out with buffer solution followed by SERS measurement. Target antigen biomarkers are quantitatively assayed by monitoring the intensity change of characteristic SERS peak of report molecules labeled on the surface of metal nanoparticles.13 Although this aptamer-based strategy greatly improved the selectivity of SERS detection, it has several drawbacks such as requirement of long incubation time in each binding step, loss of biological activity resulting from exposure to air, and poor reproducibility of SERS signal.15 To resolve these problems, Yoon et al.11 developed a method for detection of thrombin using a SERS-based magnetic aptasensor. They used aptamer conjugated-magnetic beads as capturing probes and separation tools instead of a solid substrate, which overcame problems of slow reaction between aptamer and thrombin. However, both Raman active molecules and aptamers were physically attached to the metallic nanoparticles, which may lead to the dissociation of Raman active molecules in sample solution and interference of other molecules absorbing on the surface of metal nanoparticles in the complex matrixes. Moreover, the applicability of quantitative determination was not conducted in real samples. A common way to protect Raman report molecules from dissociation is to coat metal NPs with a thin shell such as polymers,16,17 transition-metal materials,15,18,19 carbon,20,21 and mesoporous silica.13,22 However, poor biocompatibility or requirement for tedious modification steps impedes these materials for further application. Chitosan (CS), the N-deacetylated derivative of chitin, is a well-known biopolymer with positively charged physicochemical and biocompatible characteristics.23 Abundant amino groups of chitosan not only make it biocompatible with the organism but also facilitate it to biofunctionalize with proteins or amino-modified aptamers.24−27 In the present study, by introducing aptamer and chitosan to improve selectivity and biocompability, we proposed an aptamer recognition induced target-bridged strategy for protein assay using aptamer modified Ag@CS NPs and aptamer (or antibody) modified MCS. The strategy relies on the availability of a pair of aptamers that bind to different regions of the protein. To demonstrate the universality, we investigated three different binding modes of the strategy taking thrombin, PDGF BB, and lgE as model proteins. To explore the feasibility of the proposed strategy, it was employed for detection of PDGF BB in human serum samples. The proposed strategy has the potential of recognizing the targets in complex matrixes and is convenient for rapid analysis of clinical samples with good selectivity, biocompability, and high sensitivity.

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EXPERIMENTAL SECTION Materials and Reagents. Silver nitrate (≥99.8%), ferric trichloride (≥99.0%), sodium acetate (≥99.0%), and ethylene glycol (≥99.7%) were purchased from Guangzhou Chemical Reagent Factory (Guangzhou, China). Glutaraldehyde (25%, v/v) was purchased from Fuchen Chemical reagent Factory 11040


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removed. Afterward, the target aptamer complexes were collected on the silicon wafer with a magnetic bar for SERS assay (Figure 1C). Application to Analysis of PDGF BB in Human Serum. To explore the applicability of the proposed strategy in protein analysis, six human serum samples of three healthy persons and three cancer patients were assayed. All of the Raman spectra reported here was collected for 10 exposure seconds in the range of 800−1500 cm−1. For comparison, the serum samples were measured by a commercial ELISA kit with a microplate reader. The procedures exactly followed the protocols suggested by the commercial kit. The optical density at the wavelength of 450 nm was detected. Control experiments were carried out to test the selectivity of this strategy. The SERS signals for negative control samples were measured at six groups of replacing PDGF BB with BSA, HRP, lgG, thrombin, lgE and replacing report TBA35 with control TBA35.

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

RESULTS AND DISCUSSION In this study, we have developed an aptamer recognition induced target-bridged strategy using MCS and Ag@CS NPs for sensitive and selective detection of protein. The strategy relies on the availability of a pair of aptamers that bind to different regions of the protein. Capture aptamers (or antibodies) were first conjugated with MCS and used for recognizing protein, and then sensing aptamers linking with 4ATP-embeded Ag@CS NPs were added for the formation of sandwich complexes of aptamer (antibody)/protein/aptamer. Once sandwich complexes were formed, the concentration of protein was indirectly reflected by Raman intensity of 4-ATP. The schematic diagram of the sandwich-type assay is shown in Figure 1. Preparation and Characterization of Sensing Probes and Capture Probes. Taking advantages of biocompatibility and abundant amino groups for further modification of CS, we prepared aptamer modified Ag@CS NPs as sensing probes and aptamer (or antibodiy) modified MCS as capture probes. Preparation and Characterization of Aptamer ModifiedAg@CS NPs. It is well-known that silver has a larger optical cross section and is more inexpensive in comparison with that of gold. However, silver is susceptible to corrosion and easily affected by ambient factors, thereby weakening plasmonic signals and limiting its applications. In order to avoid aggregation and improve the stability of Ag NPs in the exposure of harsh environment as well as to avoid dissociation of Raman report molecule, CS was coated onto the surface of Ag NPs after 4-ATP attached to Ag NPs. Figure 2 shows the TEM images of Ag@CS NPs prepared with CS of different concentrations. The thickness of CS layer on the surface of Ag NPs increased gradually with the increase of CS concentration from 0.5 mg/mL to 4.0 mg/mL. As the CS concentration increased, UV−vis absorption bands slightly redshifted (Figure S1A), which further demonstrated the increase of the thickness as the increase of the concentration of CS. The concentration of CS plays a role in SERS intensity of 4-ATP. The strongest enhancement was observed at 0.5 mg/mL of CS (Figure S1B). A slight decrease of SERS intensity took place with the concentration changing from 1.0 to 2.0 mg/mL and an obvious decrease was observed as the concentration of CS increased to 4.0 mg/mL. Considering that thicker CS coating on Ag NPs took advantage in protecting 4-ATP from diffusion, we chose 2.0 mg/mL of CS in the following experiment. To

shaker for overnight at room temperature. After that, the mixture was centrifuged and washed twice. Followed by addition of 0.1% BSA in Tris-HCl buffer and stirred for 1 h to block nonspecific adsorption. The precipitate was washed twice and redispersed into 1 mL of Tris-HCl buffer. The asprepared TBA-Ag@CS NPs were stored at 4 °C before use. Preparation of Aptamer (or Antibody)-Conjugated MCS. MCS was synthesized by the hydrothermal method.29 Briefly, sodium acetate and CS were added into the mixture solution of ferric trichloride and ethylene glycol followed by stirring for 30 min. The whole mixture was transferred into Teflon-lined stainless-steel autoclave and reacted at 200 °C for 12 h. Approximately 2.0 mg of MCS was washed three times with Tris-HCl buffer before they were mixed with 1.0 mL of 2.5% glutaraldehyde solution in Tris-HCl buffer. The mixture was set in a shaker for 2 h at room temperature. The MCS was collected with a magnetic bar and washed three times with TrisHCl buffer. Then aptamer (TBA15, TBA29, or TBA35) or antilgE antibody was added to the MCS with a final volume of 1.0 mL. The mixture was kept overnight in a shaker at room temperature. After that, the mixture was centrifuged and washed twice. This was then followed by addition of 0.1% BSA in Tris-HCl buffer and stirred for 1 h to block nonspecific adsorption. The aptamer-functionalized MCS were collected by a magnetic bar and washed twice with 1.0 mL of Tris-HCl. The final product was stored in 1 mL of Tris-HCl at 4 °C (Figure 1B). Surface-Enhanced Raman Scattering Detection. In the first step, various concentrations of thrombin, PDGF BB, and lgE solution were prepared with Tris-HCl containing 0.1% BSA, respectively. Then 25 μL of protein solution was added to 25 μL of corresponding TBA-MCS or antibody-MCS and incubated for 30 min at room temperature. The resultant MCS were further reacted with 25 μL of TBA-Ag@CS NPs for 1 h, and the sandwich aptamer complexes formed were subsequently isolated by applying a magnet to the wall of the microtube. The complexes were washed three times with 200 μL of Tris-HCl buffer using a micropipet. In this way, nonreacted reagents in residual solution were effectively 11041


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(Figure S2). It depicted that Ag@CS NPs demonstrated significantly improved SERS stability in ionic circumstances. The amino groups of CS provide abundant amino group sites for covalent linkage of biomolecules such as amino-modified aptamers and antibodies to Ag@CS NPs and MCS via glutaraldehyde. To demonstrate whether aptamer was linked with Ag@CS NPs, IR spectra and ζ-potentials value were measured (Figure S3). On one hand, after modification of Ag@ CS NPs with glutaraldehyde, the peaks 3411 and 3455 cm−1 (vNH2) became weak and wide (Figure S3A). The fact that 1588 and 1624 cm−1 (δ-NH2) disappeared while 1636 cm−1 (v−C N) appeared could be the excellent proof of cross-linking. Comparing curves c, d, e, and f with curve b, 1636 cm−1 (v− CN) becoming evident and stronger demonstrated that aptamer was successfully conjugated with Ag@CS NPs. On the other hand, the ζ-potential was also performed (Figure S3B). The ζ-potential of Ag NPs was −30.4 mV mainly due to negative charged citrate ions on the surface of Ag NPs. After coating Ag NPs with CS, the ζ-potential changed positive. After modification with aptamer, the ζ-potential turned negative, which demonstrated that Ag@CS NPs were successfully modified with aptamer. Preparation and Characterization of Aptamer (or Antibody) Modified-MCS. The MCS was synthesized through a solvothermal method. CS was used in the reaction system serving as both a ligand and a surface modification agent. Figure 3A shows representative SEM and TEM images of MCS, respectively. It is observed that the diameter of the spherical MCS is about 200 nm. Figure 3B depicts that the saturation magnetization of the MCS was 35.5 emu/g, which was enough

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, and (D) 4.0 mg/mL.

investigate the stability of Ag@CS NPs, SERS performance of Ag@CS NPs and naked Ag NPs were measured and compared in the presence of NaCl salt solution of different concentrations

Figure 3. (A) TEM image and SEM image of MCS, (B) magnetization curve of MCS, (C) XRD pattern of MCS, and (D) FT-IR spectra of CS and MCS. 11042


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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.

sandwich aptamer complexes, thus no SERS signal was observed (Figure 4B). In this approach, the analyte was sandwiched by a pair of aptamers, one capture aptamer and the other report aptamer. Capture aptamers were immobilized on the surface of MCS, while report aptamers were conjugated with 4-ATP embedded Ag@CS NPs as signaling moieties. In consideration of various combination styles between protein and binding aptamer, three different kinds of protein including thrombin, PDGF BB, and lgE were investigated. Figure 5 depicted three modes of the aptamer recognition induced target-bridged strategy.

for magnetic separation with a magnet bar. The XRD pattern of MCS is shown in Figure 3C, which can be indexed to Fe3O4. Figure 3D presents the FT-IR spectra of CS and MCS. In addition to characteristic bands of CS observing at 3435 cm−1 (v−O−H and v−N−H), 1624 cm−1 (δ−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 appearing in MCS, which indicated that CS was modified successfully with Fe3O4. In order to modify MCS with aptamer (or antibody), MCS was first functionalized with glutaraldehyde. Residual aldehyde groups reacted with amino groups of aptamer or anti-lgE antibody. FTIR spectra of MCS, glutaraldehyde modified-MCS, TBA15-MCS, TBA29-MCS, TBA35-MCS and anti-lgE antibody-MCS were performed to demonstrate the modification process (Figure S4). Compared curve a and b, after functionalization of MCS with glutaraldehyde, the peak 1625 cm−1 (δ-NH2) disappeared while 1705 cm−1(v-CHO) and 1635 cm−1 (v-CN) appeared, which was the excellent proof of modification of glutaraldehyde. We observed that the peaks 1705 cm−1(v-CHO) disappeared and 1635 cm−1 (v-CN) became stronger in curve c, d, e and f, which indicated that aptamer and anti-lgE antibody were modified onto MCS successfully. Development of the aptamer recognition induced target-bridged strategy for protein detection. Figure 4 shows the overall steps for the formation of sandwich aptamer complexes based on the aptamer recognition induced targetbridged strategy. First, aptamer modified MCS selectively recognized and captured proteins benefiting from biocompatibility of CS and affinity of the aptamer. After the aptamer conjugated Ag@CS NPs were added, the sandwich complexes of aptamer/protein/aptamer were formed based on aptamer− antigen−aptamer interactions. These complexes were isolated by a magnetic bar, washed, and redispersed in Tris-HCl buffer, and the residual Ag@CS NPs in the solution failing to form aptamer complexes were eventually removed by the washing step. Strong SERS signals of 4-ATP were observed due to the SERS effect of Ag NPs in the exposure of the laser (Figure 4A). However, in the absence of protein, it failed to form the

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

Detection of Thrombin. In most cases, capture and report aptamers of proteins have different nucleic acid sequences such as thrombin, which has one 15-mer DNA aptamer binding to the fibrinogen-recognition exosite and the other 29-mer DNA aptamer binding to the heparin-binding exosite. It is common to fabricate a sandwich complex of TBA15/thrombin/TBA29 for thrombin assay. In consideration of two different aptamer binding sites of thrombin, we compared different binding modes of aptamers and NPs, that are TBA15-Ag@CS/ 11043


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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 the 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).

thrombin/MCS-TBA29 and TBA29-Ag@CS/thrombin/MCSTBA15. It was found that the SERS signal of TBA29-Ag@CS/ thrombin/MCS-TBA15 is stronger than that of TBA15-Ag@ CS/thrombin/MCS-TBA29 (Figure S5). For the purpose of high sensitivity, therefore, it is preferable to modify Ag@CS NPs with one aptamer of higher affinity and modify MCS with the other. Detection of PDGF BB. Specifically, in limited cases, some proteins such as PDGF BB are dimeric molecule and contain two identical aptamer binding sites, thus allowing the use of a single aptamer for the sandwich formation. Taking PDGF BB as an example, TBA35 was first immobilized onto Ag@CS NPs and MCS, respectively. Excess TBA35-MCS bound with PDGF BB through one binding site and selectively captured PDGF BB from solution through protein−aptamer interaction. When TBA35-Ag NPs were added, it linked with PDGF BB through the other binding site, thus the sandwich complex of aptamer/ PDGF BB/aptamer was formed. Detection of Immunoglobulin E. Also of note, in cases when there are no two aptamers sharing identical or overlapping binding sites on protein such as lgE, it is possible to use an antibody as the second “aptamer” to form a sandwich complex of aptamer/protein/antibody. Human IgE has two different binding sites of TBA37 and antibody. For the assay, goat antihuman IgE antibody was immobilized onto the surface of MCS and TBA37 was modified onto Ag@CS NPs respectively through the link of glutaraldehyde. After human

IgE was added, human IgE bound with its antibody by the antigen−antibody interaction through one binding site. TBA37Ag NPs were attached to lgE to form sandwich of aptamer/lgE/ antibody through the other binding site. With the proposed approach, different proteins can be selectively measured through changing binding aptamers (or antibody) on the surface of Ag@CS NPs and MCS. Reproducible SERS spectra of 4-ATP with strong signal were obtained from detection of different proteins in Figure 5. Both of them show characteristic of ∼1077 cm−1, ∼1176 cm−1, and ∼1576 cm−1, which are assigned to the stretching vibration of C−S, bending vibration of C−S, and stretching vibration of C− C, respectively. Therefore, the labeled approach for protein detection overcomes the problem of poor selectivity and sensitivity occurring in label-free method. Moreover, it solves the problem of diffusion-limited kinetics on the solid substrate and can be conveniently operated with a magnetic bar. Application for PDGF BB Detection in Human Serum. To explore the feasibility of the proposed strategy, the detection of PDGF BB in human serum sample was performed. PDGF BB is a well-characterized growth factor displaying potent biological effects on angiogenesis.30 Recent studies reveal that overexpression of PDGF BB within tumors results in increased pericyte coverage, suggesting that PDGF BB signaling is also essential for tumor growth and the cancerous pericyte recruitment process.31 Therefore, the detection of PDGF BB in 11044


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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

ELISA

samplea

determinedb (pg/mL)

added (pg/mL)

recovery (%)

determinedc (pg/mL)

relative error (%)

Serum 1

142.6 ± 7.1

−8.9

101.7 ± 1.9

109.8 ± 4.8

−7.4

Serum 3

177.8 ± 7.3

169.2 ± 8.5

5.1

Serum 4

921.6 ± 68.2

872.4 ± 62.6

5.6

Serum 5

836.2 ± 52.7

915.3 ± 48.2

−8.6

Serum 6

782.4 ± 64.2

117.0 99.5 88.6 93.1 119.3 89.7 87.5 76.8 90.7 84.4 102.2 77.3

156.6 ± 6.2

Serum 2

150.0 750.0 150.0 750.0 150.0 750.0 1000 3000 1000 3000 1000 3000

726.3 ± 58.1

7.7

a Samples 1−3 were from healthy persons and samples 4−6 were from cancer patients. bQuantitative data was integrated at 1077 cm−1. The data were from three independent experiments. cThe data were collected from three independent experiments.

Table 2. Comparison of Different Optical Aptasensors for PDGF-BB Determination methods colorimetric fluorescence

chemiluminescent

SERS a

probes

LOD (pmol/L)

linearity range (nmol/L)

ref

Apt and Au NPs Apt and Ag NPs Apt and Ag NCs FAM-Apt, GO Apt-Ag NPs Apt, Au NPs, and luminol Apt, hemin, luminal, and DNAzyme FITC, luminol Au PNNs, Apt/MBA-Au NPs TBA-Ag@CS NPs and TBA-MCS

6000 200 370 167 27 60 0.68 50 0.5 0.1a

10−1000 0.2−8.2 1−50 0.167−1.167 0.2−17 0.06−6 1 × 10−3−10 0.1−100 (1 × 10−3)−(5 × 10−2) 4 × 10−4−0.2a

32 33 34 35 36 37 38 39 14 this method

The mole concentration was obtained from dividing mass concentration by molecular weight of PDGF BB.

with control TBA35, respectively (Figure S6). It showed that negligible Raman signal was obtained by replacing the proteins with BSA, HRP, and IgG, thrombin, or lgE due to the failure of the formation of a sandwich complex. Moreover, no obvious signal was obtained replacing the report TBA35 on Ag@CS NPs with control TBA35. Strong signal was obtained only when the specific protein PDGF BB and corresponding aptamer existed. The assay was very selective to PDGF BB over the tested interferences, mainly due to simultaneous recognition and high specificity between a pair of affinity aptamer probes and PDGF BB. Detection of PDGF BB in Human Serum. To test the applicability of this assay, six human serum samples collected from three healthy persons and three cancer patients were assayed by the proposed method using ELISA as the reference method. Human serum samples have been diluted 5 times with Tris-HCl buffer. It showed that concentrations of PDGF BB in the serum of cancer patients were obviously higher than that in healthy persons (Table 1). In addition, the results obtained with the proposed strategy were in good agreement with those obtained by ELISA but with a wider linear range, more convenient operation, and lower cost. Moreover, it compares favorably with those of most optical approaches14,32−39 for detection of PDGF BB (Table 2). Remarkably, the proposed aptamer recognition induced target-bridged strategy for PDGF BB detection is more sensitive and has a wider linear range than the previous SERS-based method14 probably due to bio-

human serum is significant for early diagnosis and treatment of cancer. Reproducibility. SERS spectra of the same aptamer/PDGF BB/aptamer complexes at seven different spots were collected and the intensity at 1077 cm−1 was used to test the reproducibility. The coefficient of variation was less than 8% (Figure 6A). It shows that the sandwich complexes possess good reproducibility. It is very likely that, on one hand, the CS shell showed excellent biocompatibility and protected Raman report molecules from dissociation in a harsh environment. On the other hand, the formed sandwich complexes of aptamer/ PDGF BB/aptamer were homogeneous (Figure 6B) so that reproducible signal was obtained at different spots. Sensitivity and Selectivity. Figure 6C shows SERS spectra of 4-ATP in the presence of various concentrations of PDGF BB and their corresponding calibration curves. The SERS signal of 4-ATP at 1077 cm −1 increased upon the increasing concentration of PDGF BB ranging from 10 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 was monitored and used as a quantitative evaluation of the PDGF BB antigen levels. Figure 6C (inset) shows good linear response between intensity of 4ATP and concentration of PDGF BB with a correlation coefficient of 0.9937. The limit of detection (LOD) was estimated to be 3.2 pg/mL (0.1 pmol/L). In order to test the selectivity of this strategy, six groups of contrast experiments were carried out through replacing PDGF BB with BSA, HRP, IgG, thrombin, IgE and replacing report TBA35 on Ag NPs 11045


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(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. Proc. Natl. Acad. Sci. U. S. A. 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. TrAC, 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. (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, 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. 2009, 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.

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

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CONCLUSION In the present study, we have developed an aptamer recognition induced target-bridged strategy based on Ag@CS NPs and MCS for detection of proteins. The chitosan shell and specific affinity of aptamers provide guarantees of selectivity and sensitivity of the strategy. To explore the universality of the strategy, three different kinds of proteins including thrombin, PDGF BB, and lgE as three model proteins were investigated. The strategy is suitable for detection of three kinds of proteins with two different aptamer binding sites or two identical binding sites or one aptamer binding site and another antibody binding site. To demonstrate the feasibility of the strategy, it was employed for detection of PDGF BB in human serum samples. The present SERS approach provides advantages of high sensitivity and accuracy toward PDGF BB detection, which mainly attributes to the biocompatibility of CS and specific recognition of aptamer. The LOD for PDGF BB was as low as 3.2 pg/mL and the determined results were consistent with that of the ELISA method, but with a wider linear range, convenient operation, and low cost. As more aptamer being selected for proteins, we envision that the strategy would be a universal, convenient, selective, and sensitive clinical tool for detection of proteins in complicated biological samples.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03049. UV−vis, SERS, and IR spectra and SERS intensity plots (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Phone: +86-20-84110922. Fax: +86-20-84115107. E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Major National Scientific Instrument and Equipment Development Project (Grant 2011YQ03012409) and the National Natural Science Foundation of China for financially supporting this research under Grant Numbers 21277176, 21475153, and 21127008, respectively.

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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. 11046


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Analytical Chemistry (39) Zhang, X. F.; Zhang, H.; Xu, S. X.; Sun, Y. H. Analyst 2014, 139, 133−137.

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Aptamer Recognition Induced Target-Bridged Strategy for Proteins

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