Magnetic-Encoded Fluorescent Multifunctional Nanospheres for

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Magnetic-Encoded Fluorescent Multifunctional Nanospheres for Simultaneous Multicomponent Analysis Erqun Song,*,† Weiye Han,† Jingrong Li,† Yunfei Jiang,† Dan Cheng,† Yang Song,† Pu Zhang,† and Weihong Tan‡,⊥ †

Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest University) Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing, 400715, People’s Republic of China ‡ Center for Research at Bio/nano Interface, Department of Chemistry and Department of Physiology and Functional Genomics, Shands Cancer Center, UF Genetics Institute and McKnight Brain Institute, University of Florida, Gainesville, Florida 32261-7200, United States ⊥ Molecular Science and Biomedicine Laboratory, State Key Laboratory for Chemo/Bio-Sensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, and Collaborative Research Center of Molecular Engineering for Theranostics, Hunan University, Changsha 410082, People's Republic of China S Supporting Information *

ABSTRACT: In this study, magnetic-encoded fluorescent (CdTe/Fe3O4)@SiO2 multifunctional nanospheres were constructed by adjusting the initial concentration of Fe3O4 in a fabrication process based on reverse microemulsion. The resultant multifunctional nanospheres were characterized by transmission electron microscopy, X-ray diffraction measurements, fluorescence spectrophotometry, and vibrating sample magnetometry. They showed good fluorescence properties, gradient magnetic susceptibility (weak, moderate, and strong), and easy biofunctionalization for biomolecules, such as immunoglobulin G (IgG), protein, and antibody. Then the capture efficiency of the (CdTe/Fe3O4)@SiO2 nanospheres were investigated by using the fluorophore-labeled IgG-conjugated nanospheres as a model. Further studies demonstrated the ability of these (CdTe/Fe3O4)@SiO2 multifunctional nanospheres to accomplish sequentially magnetic separation, capture, and fluorescent detection for each corresponding antigen of CA125, AFP, and CEA with a detection limit of 20 KU/L, 10 ng/mL, and 5 ng/mL, respectively, from a mixed sample under a certain external magnetic field within a few minutes. The strategy of combining magnetic-encoding-based separation and fluorescence-based detection proposed in this study shows great potential to achieve easy, rapid, economical, and near-simultaneous multicomponent separation and analysis for a variety of targets such as drugs, biomarkers, pathogens, and so on.

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successfully applied for the detection of proteins and bacteria.16−18 Although these two modes have achieved highthroughput multicomponent analysis, they still must overcome poor sensitivity and reproducibility as well as complicated operation.19−21 Therefore, it is important to develop new strategies to achieve easy, simultaneous multianalyte analysis with high sensitivity. In recent years, the development of novel nanoparticles, such as quantum dots (QDs), iron oxide magnetic nanoparticles, and multifunctional nanocomposites, has provided new opportunities to address these obstacles. On the basis of different emission wavelengths of QDs, multicolor labeling technology has been successfully applied to accomplish multiple-cell imaging22 and the detection of multiplex pathogens,23 toxins,

he accurate and simultaneous detection of multiple components, such as a series of biological targets,1 disease markers,2 or drug residuals within food,3 has attracted considerable attention based on its significance in biological research, clinical diagnosis, and food safety control. In comparison to the conventional batchwise analysis model, a strategy that achieves simultaneous detection of multiple targets from complex samples could offer several advantages, including high-throughput, short assay time, and low cost.4−7 Typically, two strategies are available for multicomponent analysis, namely, the multilabel mode and the spatial-resolved mode.6,8−10 The multilabel mode uses two or more labeled reagents, such as a radioactive element, an enzyme, fluorophores, or special metal ions, to achieve multiplex detection for disease diagnosis,11,12 environmental monitoring,13 and food safety control.14,15 The spatial-resolved mode with a single signal label can simultaneously detect different targets in different reaction areas on one substrate. For example, electrochemical and optical sensor arrays have been © 2014 American Chemical Society

Received: September 8, 2013 Accepted: September 8, 2014 Published: September 8, 2014 9434

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and DNA sequences25 with one single excitation light source, which has improved sensitivity and is easy to apply.26 Magnetic nanomaterials, especially iron oxide magnetic nanoparticles, such as Fe2O3 and Fe3O4, have attracted much attention among biomedical scientists because of their excellent magnetic manipulation and separation ability.27 Simultaneous spatially addressable sorting of multiple types of cells was achieved by labeling with different magnetic tags.28 Recently, combining fluorescent labeling with magnetic separation has attracted much attention,29,30 and significant efforts have been made to prepare fluorescent-magnetic multifunctional composites,31 such as Fe3O4−CdSe, Fe3O4@ZnS, γ-Fe2O3−CdSe, and CdSe@Fe2O3, by the covalent coupling method,32 seedinduced growth method,33 layer-by-layer approach,34 and reverse microemulsion.35,36 Indeed, the fabrication of such fluorescent-magnetic multifunctional composites has resulted in the successful separation and detection of the multiplex viruses, cells, and proteins based on multicomponent analysis strategy. For instance, newcastle disease virus and avian viral arthritis were successfully detected simultaneously by using fluorescent-magnetic multifunctional nanoparticles.37 Both leukemia and prostate cancer cells have been successfully detected and extracted from complex samples by using fluorescent-encoded, fluorescent-magnetic multifunctional nanobioprobes.38 More interestingly, Wilson and coworkers prepared multicolor fluorescent-encoded, fluorescentmagnetic composites for simultaneous analysis of three different kinds of proteins.25,39 It turns out that this technique could achieve the simultaneous detection of multiple targets; however, it could not separate out each single component and achieve further analysis from the multicomponents. On the basis of the controllability of magnetic performance, magneticencoded fluorescent multifunctional composites with different degrees of magnetism may achieve the above goal. Recently, fluorescent-magnetic, dual-encoded multifunctional bioprobes have been applied for the simultaneous separation and detection of multiple types of lectin in a complex mixed sample.40 However, in this strategy, micrometer-sized particles could complicate the interaction between targets and micrometer-sized bioprobes, a problem easily overcome by the lightness and small size of nanometer-sized bioprobes. Therefore, magnetic-encoded fluorescent-magnetic nanospheres (FMNS) with differential magnetic potentials, i.e., weak, moderate, and strong, are promising tools to achieve simultaneous separation and detection by controlling the separation of each component under an external magnetic field. In this work, we developed multimagnetic/magnetic-encoded (CdTe/Fe3O4)@SiO2 FMSN based on the reverse microemulsion method (as shown in Scheme 1). Water-soluble CdTe QDs and Fe3O4 nanoparticles were simultaneously embedded into a silica shell to fabricate FMNS by hydrolysis and condensation of tetraethylorthosilicate (TEOS) under ammonia catalysis, and different degrees of FMNS magnetism were controlled by simply adjusting the concentration of Fe3O4 initially added. Characterization of the structure and properties of (CdTe/Fe3O 4)@SiO2 FMNS demonstrates that the resultant FMNS have small size (about 60 ± 5 nm), excellent fluorescence, different magnetic potentials, and good stability. Moreover, such FMNS could be easily conjugated with immunoglobulin G (IgG), protein A, and antibody (Ab), respectively, showing easy biofunctionalization, and a high capture efficiency of each type of FMNS with different magnetic susceptibility was demonstrated by using fluoro-

Scheme 1. Schematic of Fabrication of (CdTe/Fe3O4)@SiO2 FMNS with Different Magnetic Potentials: Weak (WFMNS), Moderate (M-FMNS), and Strong (S-FMNS), Respectively, By the Reverse Microemulsion System

phore-labeled IgG-FMNS conjugates as a model. Importantly, the magnetic-encoded FMNS could achieve magnetic separation and fluorescent detection of multiplex antigens sequentially from a mixed sample under a certain external magnetic field within a few minutes. Thus, the strategy of combining magnetic-encoding-based separation and fluorescence-based detection has the potential to achieve easy, rapid, economical, and near-simultaneous multicomponent separation and analysis for toxins, drugs, biomarkers, viruses, bacteria, and mammalian cells.

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EXPERIMENTAL SECTION Materials and Apparatus. Iron(III) chloride hexahydrate (FeCl3·6H2O), iron(II) sulfate heptahydrate (FeSO4·7H2O), sodium borohydride (NaBH4), citric acid monohydrate (CA), and cyclohexane were purchased from Chengdu Kelong Chemical Reagents Factory (China). Acetone, n-hexanol, isopropyl alcohol, anhydrous ethanol, and aqueous ammonia solution (NH4OH, 25% w/w in water) were obtained from Chongqing Chuandong Chemical Co. Ltd. (China). 3Mercaptopropionic acid (MPA), cadmium chloride (CdCl2· 2.5H2O), tellurium (reagent powder), TEOS, and APTES were provided by Shanghai Jingchun Industrial Co. Ltd. (China). Protein A and T-octylphenoxypolyethoxyethanol (Triton X100) were supplied by Beijing Ding Guo Changsheng Biotechnology Co. Ltd. (China). Mouse anti-CEA antibody, mouse anti-AFP antibody, mouse anti-CA125 antibody, and poly(dimethyldiallyl ammonium chloride) (PDDA, average Mw 100 000−200 000 (low molecular weight), 20 wt % in H2O) was obtained from Sigma-Aldrich Co. Ltd. (United States). Fluorescein (FITC)-goat antirabbit IgG and phycoerythrin (PE)-goat antirabbit IgG were provided by ProteinTech Group. Quantum dot (QD615 nm)-labeled IgG was provided by our laboratory. FITC-CEA, Cy3-AFP, and PE-Cy5-CA125 were purchased from Shanghai Haochen Biotechnology Co. Ltd. 9435

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Conjugation of (CdTe/Fe3O4)@SiO2 FMNS with Fluorscently Labeled IgG Molecules. First, 0.2 mL of 1% glutaraldehyde solution was added to 0.5 mL of the above prepared amino-modified FMNS suspension (2.0 mg/mL), and the mixture was shaken for 0.5 h at 37 °C, followed by washing with PBS three times to remove unreacted glutaraldehyde. Then the products were dispersed in 0.5 mL of PBS and reacted with 100 μL of fluorescently labeled goat antirabbit IgG molecules (FITC-IgG, PE-IgG and QD615 nm-IgG) (10 μg/ mL), respectively) for 2 h at 37 °C with continuous shaking.41 Subsequently, the resulting mixtures were washed with PBS several times to remove superfluous IgG molecules. Finally, IgG-FMNS including FITC-IgG-conjugated W-FMNS (WFMNS@IgG-FITC), PE-IgG-conjugated M-FMNS (MFMNS@IgG-PE), and QD615 nm-IgG-conjugated S-FMNS (SFMNS@IgG-QD615 nm) were obtained and stored in PBS at 4 °C respectively. Preparation of Ab-Modified (CdTe/Fe3O4)@SiO2 FMNS Nanoprobes. The Ab-modified (CdTe/Fe3O4)@SiO2 FMNS nanoprobes (Ab-FMNS) were prepared by an indirect strategy. First, NH2-FMNS nanospheres were reacted with protein A as the similar procedure as NH2-FMNS conjugating with fluorescently labeled IgG molecules. That is, after the FMNSNH2 nanocomposites modified with glutaraldehyde, the products were dispersed in 0.4 mL of PBS and reacted with 100 μL of protein A (500 μg/mL) for 2 h at 37 °C with continuous shaking, followed by washing with PBS several times to remove protein A molecules. Then the protein A modified FMNS (protein A-FMNS) reacted with antibodies (anti-CEA Ab, anti-AFP Ab, and anti-CA125 Ab) in 0.4 mL of PBS for 2 h at 37 °C with continuous shaking according to a previous work, respectively.42 After fully washing with PBS, the Ab-FMNS nanoprobes (W-FMNS@anti-CEA-Ab, M-FMNS@ anti-AFP-Ab, and S-FMNS@ anti-CA125-Ab) were finally obtained and stored in PBS at 4 °C. Magnetic Capturing and Separating of IgG-FMNS Conjugates. W-FMNS@IgG-FITC, M-FMNS@IgG-PE, and S-FMNS@IgG-QD615 nm conjugates were each placed on a magnetic scaffold, which is a magnetic particle concentrator consisting of two pieces of Nd−Fe−B magnets with a plastic shell (with surface magnetic strength about 0.4 T). Then, the fluorescence intensity of each conjugate was measured with the elapse of time to determine the capture efficiency of each type of IgG-FMNS. Magnetic Separating and Detecting of Multiplex Antigens with Ab-FMNS Nanoprobes. Three different kinds of fluorescently labeled antigens (FITC-CEA, Cy3-AFP, and PE-Cy5-CA125) were first mixed and incubated in a microplate well coating with capturing Abs for CEA, AFP, and CA125, respectively, and then the three types of Ab-FMNS nanoprobes (W-FMNS@anti-CEA-Ab, M-FMNS@anti-AFPAb, and S-FMNS@anti-CA125-Ab) were added into the same well. After full incubation and washing, the whole system was treated with dissociation buffer and subjected to magnetic capture and separation as done for the IgG-FMNS mixture described above. Then each captured constituent was spread on a glass slide and imaged under an inverted fluorescence microscope.

(China). All chemicals used were of analytical reagent grade, and no further purification was required. The water used in this study was prepared using a Milli-Q water purification system (18.2 MΩ, Elga, England). The fluorescence spectra were obtained using a fluorescence spectrophotometer (F-7000, Hitachi). The structure of CdTe, Fe3O4, and (CdTe/Fe3O4)@SiO2 was determined by X-ray diffraction (XRD) measurement (X-7000, Shimadzu Corporation) at room temperature. Morphology and microscopic structure were characterized using a transmission electron microscope (TEM) (LIBRA 200PE, German Carl Zeiss Company). The magnetism of Fe3O4 nanoparticles and (CdTe/Fe3 O4 )@SiO 2 nanospheres was measured by a vibrating sample magnetometer (VSM) (LDJ-9600, Microsence Corporation). Infrared spectra of all products were characterized using Fourier transform-infrared spectroscopy (FT-IR) (Spectrum GX FT-1730, PerkinElmer). The zeta potential and dynamic light scattering (DLS) size distribution was measured using a Malvern Zetasizer 3000 HS. Fluorescent images were obtained under an inverted fluorescence microscope (Olympus IX71). A TB246 magnetic scaffold was used to achieve the magnetic separation and capture. (Promega, Beijing Biotech Co. Ltd.). Preparation of (CdTe/Fe3O4)@SiO2 FMNS with Different Degrees of Magnetism. After Fe3O4 and CdTe nanoparticles were successfully prepared (the details of preparation were described in Supporting Information), multimagnetic (CdTe/Fe3O4)@SiO2 FMNS were fabricated by reverse microemulsion, as schematically outlined in Scheme 1. First, 7.5 mL of cyclohexane, 1.8 mL of Triton X-100, and 1.6 mL of n-hexanol were added in a flask under stirring. After 0.5 h, 0.8 mL of CdTe solution (20 μmol/L) and 50 μL Fe3O4 solution (10 mg/mL) were added into the above mixture to form a water-in-oil reverse microemulsion of water solution/ TritonX-100/n-hexanol/cyclohexane. The multimagnetic (CdTe/Fe3O4)@SiO2 FMNS could be obtained by adjusting the concentration of Fe3O4 initially added. Subsequently, 0.1 mL of PDDA solution (0.075%, v/v) and 0.1 mL of TEOS were added to the above solution and stirred for another 20 min. Next, 0.12 mL of aqueous ammonia solution (NH4OH, 25% w/w in water) was added to initiate TEOS hydrolysis in the dark. After reaction for 24 h, 20 mL of acetone was added to the solution to break the microemulsion system, and the resulting mixture was centrifuged (60g × 5 min). The precipitate was redispersed in isopropanol and then washed in sequence with anhydrous ethanol and water. As a result, a set of magneticencoded (CdTe/Fe3O4)@SiO2 FMNS was prepared, including (CdTe/Fe3O4)@SiO2 FMNS with weak, moderate, and strong magnetic potentials, abbreviated as W-FMNS, M-FMNS, and SFMNS, respectively. Preparation of Amino-Modified (CdTe/Fe3O4)@SiO2 FMNS. (CdTe/Fe3O4)@SiO2 FMNS were modified with an amino group for further application. Briefly, 0.5 mL of (CdTe/ Fe3O4)@SiO2 FMNS (2.0 mg/mL) was dissolved in a mixture of 5 mL of anhydrous ethanol and 0.16 mL of ultrapure water with stirring for 10 min. Then 0.1 mL of APTES was added dropwise to the above solution and reacted at 40 °C for 2 h under continuous stirring. The final product (NH2−FMNS) was washed successively with anhydrous ethanol, ultrapure water, and phosphate buffer solution (PBS, 0.01 mol/L, pH 7.4).

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RESULTS AND DISCUSSION Water-in-oil (W/O) microemulsions (water droplets dispersed in bulk oil), also termed reverse microemulsions, have been found extremely useful as nanoreactors for the confined 9436

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Figure 1. TEM images of (CdTe/Fe3O4)@SiO2 FMNS: (A) W-FMNS, (B) M-FMNS, and (C) S-FMNS.

synthesis of nanoscale nanocomposites having diverse sizes, shapes, and functions.43 In the reverse microemulsion strategy, the core precursors are usually clad by an outside silica shell which is nontoxic, relatively stable in biological environments, and conducive to further applications.44 In this study, multimagnetic magnetic-encoded (CdTe/Fe 3 O 4 )@SiO 2 FMNS were prepared by using the W/O reverse microemulsion strategy, as shown in Scheme 1. In this reaction system, water-soluble CdTe QDs and CA-Fe3O4 (Their pattern was characterized by FT-IR and TEM, as shown in Figure S1A,B in the Supporting Information.) served as the water phase, followed by addition to the TritonX-100/n-hexanol/ cyclohexane mixture to form a stable reverse microemulsion system. Afterward, the whole system was encapsulated in a silica shell by hydrolysis and condensation of TEOS under ammonia catalysis to obtain the (CdTe/Fe3O4)@SiO2 FMNS. (CdTe/Fe3O4)@SiO2 FMNS with three distinct degrees of magnetic potential (W(eak)-FMNS, M(oderate)-FMNS, and S(trong)-FMNS) could be obtained by adjusting the initial concentration of Fe3O4. The structure and properties of (CdTe/Fe3O4)@SiO2 FMNS were demonstrated as follows, Size and Structure Characterization of (CdTe/Fe3O4)@ SiO2 FMNS. As illustrated by a series of representative TEM images shown in Figure 1, (CdTe/Fe3O4)@SiO2 FMNS showed uniform spherical patterns and smooth surfaces with an average diameter of 60 nm. Images also clearly show that the FMNS contained two different sizes of nanoparticles: the slightly larger and darker spherical nanoparticles with average diameter of 12 nm and the smaller and lighter spherical nanoparticles with an average diameter of 3.2 nm. XRD patterns of the resultant FMNS (Figure 2) showed the distinguishing diffraction peaks of SiO2 nanospheres (a strong and broad peak around 2θ = 20−25°), Fe3O4 (peaks at 2θ = 30°, 35°, 43°, 53°, 57°, and 62°), and CdTe QDs (peaks at 2θ = 25°, 40°, and 47°), respectively, confirming that the nanospheres, 12 and 3.2 nm of spherical nanoparticles, as shown in Figure 1, were silica nanospheres, Fe3O4 magnetic nanoparticles, and CdTe QDs, respectively. The gradually increasing number of Fe3O4 magnetic nanoparticles in the silica nanospheres corresponds to the gradually increasing initial concentration of Fe3O4 (Scheme 1). Overall, these results proved that (CdTe/Fe3O4)@SiO2 FMNS with different amounts of Fe3O4 magnetic nanoparticles were successfully prepared based on a W/O reverse microemulsion strategy. Characterization of (CdTe/Fe3O4)@SiO2 FMNS Properties. The fluorescence and magnetic properties of (CdTe/ Fe3O4)@SiO2 FMNS were further investigated. As shown in Figure 3A, no obvious change of the fluorescence emission peak of FMNS was observed, and their fluorescence intensity remained strong (inset photographs in Figure 3A) for further use, even though the fluorescence intensity decreased gradually

Figure 2. XRD patterns of Fe3O4, CdTe QDs, and (CdTe/Fe3O4)@ SiO2 FMNS.

as the concentration of Fe3O4 increased. The slight blue shift might have resulted from the shedding of thiol ligands from the surface of QDs, leaving the QDs unprotected.45 According to a previous study, the decreased fluorescence intensity of FMNS could be ascribed to the relative amount of CdTe QDs in the FMNS which gradually reduces with increasing concentration of Fe3O4, as shown in Figure 1. In the alternative, such decreased fluorescence of FMNS could have been influenced by the absorbance of Fe3O4 nanoparticles.46 The magnetic property of the (CdTe/Fe3O4)@SiO2 FMNS was investigated using a VSM at 300 K. The magnetic hysteresis loops of magnetic-encoded W-FMNS, M-FMNS, and S-FMNS are shown in Figure 3B. The zero coercivity and zero remanence shown in Figure 3B indicated that all the (CdTe/ Fe3O4)@SiO2 FMNS maintained superparamagnetism equal to that of pure Fe3O4 magnetic nanoparticles (Figure S1C in the Supporting Information). The average magnetic saturation (Ms) values of W-FMNS, M-FMNS, and S-FMNS were 1.67 emu/g, 9.47 emu/g, and 15.28 emu/g, respectively, which could be ascribed to the increased amount of Fe3O4 in the silica nanospheres, as shown in Figure 1 and Figure S2 in the Supporting Information. Moreover, the three Ms values were distinctly different from each other, implying that these FMNS with different magnetic potentials could all be sequentially separated from each other under certain magnetic fields. This hypothesis was further proved by the different magnetic response of FMNS to the external magnetic field generated by a magnetic scaffold, as defined previously. As shown in Figure 3C, after being placed in the magnetic scaffold, most SFMNS were quickly attracted to the tube wall within 40 s, and 9437

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Figure 3. Fluorescence spectra (A) and magnetic hysteresis loops (B) of (CdTe/Fe3O4)@SiO2 FMNS at room temperature. The insets in (A) are photographs of (CdTe/Fe3O4)@SiO2 FMNS under UV light. (C) Photographic images of magnetic attraction process (C) of the (CdTe/Fe3O4)@ SiO2 FMNS with different magnetic potentials taken under UV light at different time points (0s, 40s, 100s and 240s). (a) W-FMNS, (b) M-FMNS, and (c) S-FMNS.

Figure 4. Fluorescence emission spectra of (A) W-FMNS@IgG-FITC, (B) M-FMNS@IgG-PE, and (C) S-FMNS@IgG-QD615 nm. (D) Capture efficiency of each fluorescently labeled IgG-conjugated FMNS: W-FMNS@IgG-FITC, M-FMNS@IgG-PE, and S-FMNS@IgG-QD615 nm at different times.

Bioconjugation of (CdTe/Fe3O4)@SiO2 FMNS. With good fluorescence-magnetic properties and stability, the asprepared magnetic-encoded (CdTe/Fe3O4)@SiO2 FMNS were subjected to biofunctionalization with IgG and Abs. The capture efficiency and further applications for multiplex capture and detection of FMNS were investigated by using three different kinds of fluorescently labeled IgG (FITC-IgG, PE-IgG, and QD615 nm-IgG) and antigen (FITC-CEAA, CY3-AFP, and CY5-CA125) as model molecules, respectively. Before conjugation with IgG or Ab molecules, the (CdTe/Fe3O4)@ SiO2 FMNS were first modified with APTES to generate an amino group, which was confirmed by the FT-IR spectra and zeta potential, as shown in Figures S7 and S8 in the Supporting Information. Then, FITC-IgG, PE-IgG, and QD615 nm-IgG were conjugated with NH2−FMNS through a coupling reaction between the amino groups (from FMNS and IgG molecules) and aldehyde group of glutaraldehyde and characterized by fluorescence spectra. As shown in Figure 4A, an obvious emission peak at 520 nm, the emission peak of FITC, appears together with the emission peak of 550 nm, the emission peak

the bulk solution became clear, while no obvious collection was noted for either M-FMNS or W-FMNS. However, after 100 and 240 s, respectively, M-FMNS and W-FMNS were magnetically captured at nearly the same degree as that of the captured S-FMNS at 40 s. These results again indicate that the three types of as-prepared magnetic-encoded FMNS could be separated sequentially based on their differential magnetic response density under a certain magnetic field. Therefore, all of the above results demonstrated that the magnetic-encoded (CdTe/Fe3O4)@SiO2 FMNS have been successfully constructed, providing a working platform for successive separation and simultaneous detection of multicomponents. The stability of (CdTe/Fe3O4)@SiO2 FMNS was also investigated by studying the effects of different buffer solutions and storage time on their fluorescence intensity, magnetic response under external magnetic field and DLS size distribution. The results showed in Figures S3−S6 in the Supporting Information suggest good stability of (CdTe/ Fe3O4)@SiO2 FMNS. 9438

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240 s, respectively. Therefore, these results showed the distinct magnetic response potentials of FMNS under certain magnetic fields, indicating specific magnetic separation and detection for multiple targets from a complex sample. To demonstrate that these FMNS could still achieve selective magnetic separation, capture, and fluorescent detection for multicomponents from a complex sample, each type of IgGFMNS conjugates was magnetically captured and fluorescently detected, sequentially, from the ternary mixture of W-FMNS@ IgG-FITC, M-FMNS@IgG-PE, and S-FMNS@IgG-QD615 nm, as illustrated in Scheme 2.

of FMNS, suggesting the successful conjugation of W-FMNS with FITC-IgG (W-FMNS@IgG-FITC). Figure 4B,C also showed the simultaneous maximum emission peak of fluorophores (the emission peak of PE and QD615 nm at 575 and 615 nm, respectively) and the emission peak of FMNS at 550 nm, indicating the production of M-FMNS@IgG-PE and S-FMNS@IgG-QD615 nm conjugates. For the conjugation with Abs, NH2-FMNS nanospheres were prereacted with protein A (protein A-FMNS) based on the same principle as NH2-FMNS coupled with IgG above as illustrated in Figure S9A in the Supporting Information. Protein A here was used to sitedirected immobilization of Abs when they were coupled to the surface of FMNS nanospheres to improve the likelihood of antigen−antibody interactions.47 As protein A could bind Abs through interaction with their Fc region specifically, the successful bind of protein A with NH2-FMNS was proved by using FITC labeled antibodies (FITC-Abs) reacting with protein A-FMNS. The FITC-Abs will bind specifically to the FMNS, enabling them to produce green fluorescence under blue light excitation due to the FITC. As shown in Figure S9 in the Supporting Information, when compared with a control experiment (experiment was carried out as above except without adding glutaraldehyde and protein A), bright green fluorescence dots were obtained under florescence microscopy while there is not any green fluorescence dot in the control group except the red fluorescence dots from FMNS nanospheres themselves. These results demonstrated the successful couple of protein A with FMNS nanospheres. Then Abs were linked to protein A-FMNS through interaction of protein A with their Fc region. Magnetic Capture Efficiency Analysis of IgG-FMNS. The magnetic capture efficiencies of the magnetic-encoded WFMNS, M-FMNS, and S-FMNS were investigated by using three different kinds of fluorescently labeled IgG(FITC-IgG, PE-IgG, and QD615 nm-IgG) conjugated FMNS as a model. The magnetic capture for IgG-FMNS proceeded under the external magnetic field produced by the same magnetic scaffold as shown in Figure 3C. Since the fluorescence intensity of the suspensions is positively correlated with the concentration of FMNS in the suspensions, the capture efficiencies of each type of IgG-FMNS could be calculated by eq 1: Ec = (1 − FT/F0) × 100%

Scheme 2. Schematic of Sequential Magnetic Capture and Separation for Each Type of FMNS with Captured Targets from the Mixture under an External Magnetic Field

In the ternary mixture, Figure 5A shows that the fluorescence emission peaks of FITC, PE, QD615 nm, and FMNS at 520, 575, 615, and 550 nm, respectively, appeared simultaneously at the beginning of magnetic operation. The fluorescence intensity of a certain emission peak gradually decreased with increased magnetic capture time. Specifically, the emission peak of QD615 nm first disappeared after 40 s of magnetic capture, indicating that S-FMNS@IgG-QD615 nm conjugates were first captured by the magnetic scaffold. This phenomenon could be attributed to the strong magnetic potential of S-FMNS. After 100 and 240 s, the emission peak of PE and FITC successively disappeared. Figure 5B showed the magnetic capture efficiencies of each kind of IgG-FMNS conjugates from the ternary mixture. The SFMNS@IgG-QD615 nm conjugates could be separated and captured from the mixture first with capture efficiency of ∼100% within 40 s; then, M-FMNS@IgG-PE and W-FMNS@ IgG-FITC were successively separated and captured with capture efficiency of 90.88% and 95.47% within 100 and 240 s, respectively. These differences under an external magnetic field are expected to enable (CdTe/Fe3O4)@SiO2 FMNS with differential magnetic potentials (W-FMNS, M-FMNS, and SFMNS) to be effective probes for near-simultaneous selective separation and detection of each target from complex samples Magnetic Separating and Fluorescent Detecting of Multiplex Antigens with Ab-FMNS Nanoprobes. To evaluate the feasibility of Ab-FMNS nanoprobes for magnetic capture and fluorescnec detection of real targets, the three kinds of magnetic encoded Ab-FMNS nanoprobes (W-FMNS@antiCEA-Ab, M-FMNS@anti-AFP-Ab, and S-FMNS@anti-CA125Ab) were first applied to capture and detect their corresponding single component targets (CEA, AFP, and CA125), respectively. The results shown in Figure S11 in the Supporting Information suggest that each kind of Ab-FMNS nanoprobes could detect their own targets effectively, which means that W-

(1)

where Ec is the capture efficiency of each tpye of IgG-FMNS, and F0 and FT are the fluorescence intensity of IgG-FMNS suspensions before and after magnetic capture, respectively. The amounts of each type of IgG-FMNS in suspensions were monitored by measuring the fluorescence spectra of samples at the farthest spot from the magnetic scaffold. The fluorescence spectra of the W-FMNS@IgG-FITC, M-FMNS@IgG-PE, and S-FMNS@IgG-QD615 nm in suspension (as shown in parts A, B, and C of Figure S10 in the Supporting Information, respectively) suggested that the amount of each IgG-FMNS conjugates decreased with the extension of separation time, indirectly inferring an increased amount of captured IgGFMNS. The capture efficiencies of each IgG-FMNS were calculated by eq 1 and summarized in Figure 4D. Thus, when SFMNS@IgG-QD615 nm conjugates were completely captured at 40 s, only 14.53% of M-FMNS@IgG-PE conjugates were captured, while W-FMNS@IgG-FITC barely responded at this time point (only 4.84%). On the other hand, M-FMNS@IgGPE and W-FMNS@IgG-FITC could be captured at the same degree as S-FMNS@IgG-QD615 nm by attraction for 100 and 9439

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Figure 5. Fluorescent spectra (A) and capture efficiency (B) of each type of IgG-FMNS (W-FMNS@IgG-FITC, M-FMNS@IgG-PE, and SFMNS@IgG-QD615 nm) from a mixture at different time points under an external magnetic field.

fluorescence mentioned-above with blue light excitation, the green, kelly, and red fluorescence dots should come from FITC-CEA, Cy3-AFP, and PE-Cy5-CA125, respectively, suggesting the successfully magnetic capture of Ab-FMNS for multiplex antigens simultaneous. Then the detection limits for each kind of antigen by AbFMNS from the mixture sample were studied. After the mixture of three kinds of antigens (FITC-CEA, Cy3-AFP, and PE-Cy5CA125) and Ab-FMNS nanoprobes prepared according to the procedures described in the Experimental Section, the whole system was subjected to sequential magnetic separation and fluorescence imaging as illustrated in Scheme 2, and the results are shown in Figure 7. According to Figure 7, there are two fluorescence channels for each type of target antigen: QDs channel and dye channel. The fluorescene of detected antigen for limited quantity analysis came from the dye channel while the fluorescence of Ab-FMNS nanoprobes for the qualitative confirmation assay from the QDs channel. In this study, green fluorescence QDs were embedded into S-FMNS@anti-CA125 while red fluorescence QDs were embedded into W-FMNS@ anti-CEA-Ab and M-FMNS@anti-AFP-Ab, respectively, for distinguishable fluorescent imaging. Figure 7A showed that with the increase of concentration of CA125, the red fluorescence dots from the dye channel appeared gradually until the concentration of CA125 came to 20 KU/L while the green fluorescence dots from the QDs channel were present always. In parts B and C of Figure 7, the kelly and green fluorescence dots appeared obviously when the concentrations of AFP and CEA are 10.0 ng/mL and 5.0 ng/mL, respectively, while the red fluorescence dots from the QDs channel were present always. The results shown in Figure 7 suggested that S-FMNS@antiCA125, M-FMNS@anti-AFP-Ab, and W-FMNS@anti-CEA-Ab nanoprobes could capture and detect their correspongding targets of CA125, AFP, and CEA with a detectable limit of 20 KU/L, 10.0 ng/mL, and 5.0 ng/mL, respectively, from a mixed sample under an external magnetic field. Although the targets (antigens) detected in this study were fluorescently labeled for limit quantity analysis, it does not mean that our FMNS nanoprobes could only detect targets with fluorophore-labeling. In this manuscript the fluorescently labeled antigens were just used to prove the concept of magnetically encoded multiplex separation (here it means that different fluorescence colors are corresponding to different magnetic response), which is the most important standpoint this manuscript wants to demonstrate. For further application, detection of nonfluorescent labeled multiplex targets, the FMNS nanoprobes could be first “labeled” with QDs with different fluorescence color (it means

FMNS@anti-CEA-Ab could detect CEA (top row in Figure S11 in the Supporting Information), M-FMNS@anti-AFP-Ab could detect AFP (middle row in Figure S11 in the Supporting Information), and S-FMNS@anti-CA125-Ab could detect CA125 (bottow row in Figure S11 in the Supporting Information). As the Ab-FMNS nanoprobes for singlecomponent analysis was illustrated, their capability for near simultaneous multiple components analysis was further investgated. In this study, three kinds of different fluorescently labeled antigens (FITC-CEA, Cy3-AFP, and PE-Cy5-CA125) were employed and mixed together as the multiple analytes model. After the ternary mixture (FITC-CEA, Cy3-AFP, and PE-Cy5-CA125) was incubated with three kinds of Ab-FMNS nanoprobes (here the Ab-FMNS nanoprobes prepared without adding QDs to avoid fluorescence interference from QDs when imaging) and fully washed, the whole substances were magnetic collected, followed by imaging under a fluorescence microscope. Figure 6 showed typically green (pointed by a blue solid line arrow), kelly (pointed by blue dashed line arrow), and red (pointed by blue dotted line arrow) fluorescence dots in a dark field. As there is no other substance that could emit

Figure 6. Fluorescence image of Ab-FMNS nanoprobes taken by using a blue filter after capturing their corresponding antigens. Solid line arrows pointing to CEA captured by W-FMNS@anti-CEA-Ab, dashed line arrows pointing to AFP captured by M-FMNS@anti-AFP-Ab and dotted line arrows pointing to CA125 captured by S-FMNS@antiCA125-Ab. 9440

dx.doi.org/10.1021/ac5031286 | Anal. Chem. 2014, 86, 9434−9442

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Article

Figure 7. Fluorescence images of Ab-FMNS nanoprobes after capture and magnetic separation of their corresponding targets from a mixed sample. (A) CA125 captured and detected by S-FMNS@anti-CA125-Ab, (B) AFP captured and detected by M-FMNS@anti-AFP-Ab, and (C) CEA captured and detected by W-FMNS@anti-CEA-Ab.

that QDs with different fluorescence emission wavelengths could be used to fabricate FMNS nanoprobes) to verify the separated nonfluorescent labeled targets. Actually, once the magnetically encoded multiplex separation and detection technique were successfully developed, nonfluorescent labeled targets could be differentiated just based on the different magnetic response after the capture moieties for corresponding targets conjugated with the FMNS nanoprobes. Also the amount of each target could be determined by fluorescence intensity of each FMNS nanoprobe according to the obtained linear equations, which is a work we are going to accomplish in the near future.

and fluorescent detection of each different type of targets (antigens) from mixture sample within several minutes. Taken together, such a magnetic-encoded (CdTe/Fe3O4)@SiO2 FMNS-based assay strategy exhibits the potential of the assay which is needed to accomplish near-simultaneous separation and analysis of multitargets, such as viruses, bacteria, or mammalian cells, from complex samples, which will provide a novel tool with which to improve disease diagnosis, food safety control, and environment monitoring.

CONCLUSION In summary, magnetic-encoded (CdTe/Fe3O4)@SiO2 FMNS with good fluorescence properties and differential magnetic potentials were fabricated using a simple and convenient reverse microemulsion method. The proposed magneticencoded (CdTe/Fe3O4)@SiO2 FMNS with good stability could be successfully conjugated with multiplex biomolecules, showing their biofunctionalization ability. Furthermore, the FMNS showed high magnetic capture efficiency, more than 90% for each type of FMNS with different magnetic susceptibilities (W-FMNS, M-FMNS, and S- FMNS) by using fluorescently labeled IgG-FMNS conjugates as a model. And moreover, these magnetic-encoded (CdTe/Fe3O4)@SiO2 FMNS successfully achieved sequentially magnetic separation

Experimental procedures for the preparation of CA-Fe3O4 magnetic nanoparticles and CdTe QDs, FT-IR spectrum, morphology, and magnetic properties of CA-Fe3O4 nanoparticles, the content of iron in each kind of (CdTe/Fe3O4)@ SiO2 nanospheres, the stability characterization of (CdTe/ Fe3O4)@SiO2 nanospheres, the FT-IR spectra and zeta potential of (CdTe/Fe3O4)@SiO2 before and after modification with an amino group, schematic of the preparation and fluorescent imaging validation of Ab-FMNS nanoprobes, fluorescent spectra of IgG-FMNS in suspension at different time points, and fluorescent images of Ab-FMNS nanoprobes after capturing their corresponding targets in batches. This material is available free of charge via the Internet at http:// pubs.acs.org.

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S Supporting Information *

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +862368251225. Fax: (+86)2368251225. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grants 21005064, 21477098), Fundamental Research Funds for the Central Universities (Grants XDJK2013B009, XDJK2014A020), State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University (Grant 2012009), and the Program for Innovative Research Team in University of Chongqing (2013).

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