Multifunctional Microspheres Encoded with Upconverting

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Multifunctional Microspheres Encoded with Upconverting Nanocrystals and Magnetic Nanoparticles for Rapid Separation and Immunoassays Ying Zhang,†,∥ Chunhong Dong,‡,∥ Lin Su,‡ Hanjie Wang,*,† Xiaoqun Gong,† Huiquan Wang,§ Junqing Liu,‡ and Jin Chang*,† †

Institute of Nanobiotechnology, School of Life Sciences, Tianjin University, Tianjin, 300072, People’s Republic of China Institute of Nanobiotechnology, School of Materials Science and Engineering, Tianjin University, Tianjin, 300072, People’s Republic of China § School of Electronics and Information Engineering, Tianjin Polytechnic University, Tianjin 300387, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: Immunoassays based on the downconversion target materials (organic dyes or quantum dots) lead to fairly strong spectral interference between the coded signal and reporter signal, which seriously affects the detection accuracy and hampers their applications. In this work, a new kind of upconverting nanocrystals encoded magnetic microspheres (UCNMMs) were designed and prepared successfully to solve the problem mentioned above. The UCNMMs were obtained by incorporating magnetic Fe3O4 nanoparticles and upconverting nanocrystals with polystyrene microspheres. Due to that upconverting nanocrystals (UCNs) and reporter signals are excitated by near-infrared and UV/visible light separately, immunoassays based on UCNMMs do not occur optical spectral interferences. Furthermore, these new functionalized UCNMMs have excellent properties in binding biomolecules and fast separating, which would have large potential applications in multiplexed assays. KEYWORDS: upconverting nanocrystals, magnetic nanoparticles, multifunctional microspheres, fast immunoassay, double-antibody sandwich immunoassay in bioseparations, such as immunoassays,9 cell isolation,10 virus separation, protein purification,15 DNA/RNA extraction,11,12 enzyme immobilization,13 drug magnetic targeting,14 and so on. As we know, the conventional centrifugal separation was timeconsuming, troublesome and with low recovery ratio. While, IMMS are able to be efficient, automated, parallelizable and controllable to separate the target objects. Moreover, the immunomagnetic separation (IMS) with low shearing stress has behaved gently during antigen−antibody binding and washing, so as not to break the conformation and function of protein.15−17 Furthermore, encoding microspheres with fluorophores is probably the most established method for spectral encoding. Until now, this has primarily been done with organic dye molecules or quantum dots (QDs) that embedded into or

1. INTRODUCTION Immunoassays on a molecular level, such as proteins, have been applied in bioanalysis, which is very helpful to get important information for disease diagnosis, food inspection, water environment detection, and so on.1 At present, there are some common methods that already used for protein detection such as enzyme-linked immunosorbent assay (ELISA), Western blotting, and mass spectrometry (MS) analysis.2−4 These methods have a major impact on high-density screening, but how to simultaneously achieve rapid, accurate, high-throughput detection is still a severe problem to be solved. Multiplexed suspension arrays offer advantages including flexibility in target selection, fast binding kinetics, and well controlled binding conditions that may be conducive to solve the problem mentioned above, where polystyrene microspheres play an important role as solid carriers to conjugate probe molecules.5−7 Especially, immunomagnetic microspheres (IMMS) as a new type of immunological technique has been studied at home and abroad,8 which widely have been applied © XXXX American Chemical Society

Received: October 18, 2015 Accepted: December 10, 2015

A

DOI: 10.1021/acsami.5b09913 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. TEM and upconversion photoluminescence images of six distinct UCNs: (A) blue emission UCNs (NaYF4:Yb, Tm, Er, Mn = 75/25/0.3/ 0/0); (B) sky-blue emission core−shell UCNs (NaYF4:Yb, Tm, Er, Mn = 75/25/0.3/0/0@48/12/0/1.2/0); (C) cyan emission UCNs (NaYF4:Yb, Tm, Er, Mn = 75/25/0.3/0.2/0); (D) green emission UCNs (NaYF4:Yb, Tm, Er, Mn = 80/20/0/2/0); (E) yellow emission UCNs (NaYF4:Yb, Tm, Er, Mn = 80/20/0/0.4/0); (F) orange emission core−shell UCNs (NaYF4:Yb, Tm, Er, Mn = 35/20/0/1/45); (G−H) EDX and XRD data of yellow emission UCNs, respectively.

attached to external surface of polymer or silica microbeads for capacity spectral encoding.18−20 Although immunoassays based on quantum dots encoded magnetic microspheres (QDMMs) have been studied for years,21−23 there are still some drawbacks hampered their applications. For instance, QDs could be widely excited by light in the visible and UV region, which leads to fairly strong background fluorescence.24 Hence, the ineluctable weakness of immunoassay system based on QDMMs is that both the QDs and reporter dyes can be synchronously excited by UV or Vis light. And usually the former’s signal intensity is much stronger than that of the latter, which results in fluorescent overflow and obtains an overlapped composite signal, severely limiting the detection of reporting signals, even at low reporter concentrations. In order to minimize the spectral interference and guarantee the accuracy of detection, it is necessary to separate the optical region of the coded signals from the reporter signals as far as possible. Herein, a new kind of upconverting nanocrystals encoded magnetic microspheres (UCNMMs) were designed and

prepared successfully to address the problems previously discussed. The UCNMMs encapsulate upconverting nanocrystals (UCNs) and Fe3O4 nanoparticles into the polystyrene microspheres. The coded signals of UCNs can only be excited by near-infrared (NIR) light into visible emission. Meanwhile, the reporter dyes have another excitation light source in UV or visible region, which have no absorbance in NIR domain. Thus, the detection of coded signals and reporter signals can be separated completely.25,26 Moreover, the UCNs materials exhibit great properties with sharp emission bands, high resistance to photobleaching, low toxicity, no interference from biomoleculars autofluorescence, and so an.27−31 Therefore, the immunoassay system based on the UCNMMs has unique advantages of no optical crosstalk interference between the upconversion spectral encoding and any reporter dye.31 Besides, Fe3O4 magnetic nanoparticles (MNs) have the property of superparamagnetism.32 To sum up, the synthesis of UCNMMs promotes the development of multichannel suspension arrays, while the application of magnetic microspheres realizes rapid separation and analysis.33 In this work, B


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Figure 2. (A) Schematic description of the preparation process about upconverting nanocrystals encoded magnetic microspheres (UCNMMs); (B− D) SEM images of blank microspheres,magnetic microspheres and UCNMMs; and high-resolution TEM images and line-scanning EDS datas of (E) magnetic microspheres and (F) UCNMMs.

of the hexagonal β-phase NaYF4:Yb, Er crystals.36,37 In brief, the aforementioned results clearly demonstrate that the UCNs with different elements doping ratios were synthesized successfully. 2.2. Synthesis and Characterization of UCNMMs. The encapsulation mechanism for preparing UCNMMs is shown in Figure 2A. First, magnetic microspheres were prepared by the solvent evaporation method at room temperature. Accoring to the line-scanning Energy dispersive spectrometer (EDS) analysis of magnetic microspheres in Figure 2E, the MNs tend to be wrapped by inside polymer chains close to the surface of microspheres. Then, UCNMMs were achieved by the self-healing encapsulation strategy at 180 °C, and UCNs dispersed homogeneously inside the microspheres (Figure 2F). As shown in scanning electron microscope (SEM) images (Figure 2B−D), in contrast with the empty microspheres and magnetic microspheres, the surface of UCNMMs becomes much smoother and denser after loading UCNs. The reason for this is that there exists the thermal motion and interaction of molecular chains at 180 °C, UCNs were sealed into the pores successfully. When the solution was cooled rapidly to room temperature, pores on the surface of microspheres shrank suddenly, effectively avoiding the leakage of UCNs. The highly cross-linked polystyrene microspheres were prepared according to our previously published two-step seeded copolymerization

our multifunctional microspheres combine the advantages both of MNs and UCNs with diverse emission colors, which contributes to a great potential in the rapid, accurate, multiple samples immunoassay.

2. RESULTS AND DISCUSSION 2.1. Synthesis and Characterization of the Different UCNs. The morphology was characterized by the transmission electron microscopy (TEM). The TEM and upconversion photoluminescence images of six distinct UCNs (NaYF4:Yb, Tm, Er, Mn) with different doping ratios are shown in Figure 1A−F. As seen, all these UCNs are uniform and monodispersed hexagonal β-phase nanocrystals with high crystallinity and purity after synthesis for 1 h (Figure S1). Furthermore, there are nearly no big differences about the particle sizes of the six nanocrystals with various doping elements34,35 (Figures S1 and S2). The elemental analysis and crystal structure analysis of UCNs (NaYF4:Yb,Tm,Er,Mn = 80/20/0/0.4/0) was tested by EDX and XRD. As shown in Figure 1G−H, EDX data presents the characteristic peaks of sodium (Na), yttrium (Y), fluoride (F), ytterbium (Yb), and erbium (Er) elements. The EDX data analysis reveals that the Y3+/Yb3+/Er3+ molar ratio is about 10/ 3/0.05, similar to the theoretical doping ratio. The X-ray diffraction (XRD) results illuminate that the peak positions and intensities of the nanocrystals agree well with those calculated C


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Figure 3. (A) VSM curves of different mass ratios between microspheres and MNs; (B) thermal-gravimetric (TG) and differential scanning calorimetry (DSC) curves of UCNMMs ; (C) fluorescence images of UCNMMs encoded with diverse emission colors UCNs under a 980 nm NIR laser; (D and E) fluorescence images of UCNMMs solution in cuvette under a NIR laser excitation before and after adding the extra magnetic field; and (F) bright field images of of UCNMMs solution in cuvette before and after adding the extra magnetic field.

method.38 All the microspheres samples exhibit good sphericity and uniform particle size. Calculated from the conductometric titration method, the surface-bound carboxyl content of Polystyrene microspheres was 0.4 mmol/g (Figures S4−S6). Besides, MNs were synthesized by the polyalcohol reduction method. The magnetic properties of the nanocomposites were measured including the high-resolution TEM image of Fe3O4 nanoparticles (Figure S7), vibration sample magnetism (VSM) curves of different mass ratios between microspheres and magnetic Fe3O4 nanoparticles, and thermal-gravimetric (TG) curve of UCNMMs (Figure 3A,B). Figure 3A illustrates that when the mass ratio between microspheres and magnetic Fe3O4 nanoparticles was 30:0.002/30:0.02/30:0.1/30:0.5, the saturation magnetization was increased from 0.05 to 0.3 emu/g. The results show that the specific saturation magnetization increased with the content of Fe3O4. According to Figure 3B, the thermally decomposed temperature of UCNMMs was at around 330 °C,which proves that UCNMMs own a good thermostability. The fluorescence microscopy images of UCNMMs encoded with six distinguishable emission colors UCNs are also shown in Figure 3C. After NIR excitation, the UCNMMs showed true bright colors.39 The magnetic separation speed was tested by exposing the UCNMMs solution to an external magnetic field. UCNMMs have responded strongly to the external magnetic fields in Figure

3D,F, and nearly all the UCNMMs were separated to the sidewall of the cuvette within 2 min. The narrow emission lines of UCNs allow many more codes to be accommodated in the visible domain than the quantum dots or organic dyes with broad emission bands. In general, UCNCs are constructed by a classic host material (NaYF4), doping with sensitzers (Mn2+, Yb3+) and emitters (Er3+, Tm3+). Both normalized fluorescence intensities and relative encoding intensity ratios of blue fluorescence (Tm: 450 nm, 1G4−3H4; 475 nm, 1 G4 −3 H 6), green fluorescence (Er: 525 nm, 2 H11/2−4I15/2; 545 nm, 4S3/2−4I15/2) and red fluorescence (Er: 655 nm, 4F9/2−4I15/2; Tm: 645 nm, 1G4−3F4; 695 nm,3F3−3H6) are as shown in Figure 4A. Therefore, UCNs with unique emission were created by adding the different ratios rare earth composition.28,40,41 The fluorescent stability of UCNMMs was also tested by observing the fluorescence spectra under continuous NIR illumination for different times.42 As shown in Figure 4B, the fluorescence intensity has nearly no change, suggesting that the UCNMMs behave durable photostability. Figure 4C shows the mixture of UCNMMs encoded with six different emission colors UCNs under a fluorescence microscopic field. This encoding feature of UCNs adds flexibility in designing multicolour UCNMMs for multiple detection under NIR light excitation.43 D


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Figure 4. (A) Fluorescence spectra of UCNMMs encoded with blue emission UCNs (NaYF4,Yb, Tm = 75/25/0.3) , sky-blue emission core-shell UCNs (NaYF4:Yb,[email protected](NaYF4:Yb,Er)=75/25/0.3@48/12/1.2 ), cyan emission UCNs (NaYF4:Yb, Tm, Er = 75/25/0.3/0.2), green emission UCNs (NaYF4:Yb, Er= 80/20/2), yellow emission UCNs (NaYF4:Yb , Er = 80/20/0.4) and orange emission core-shell UCNs (NaYF4:Yb, Er, Mn = 35/20/1/45) respectively and the schematic diagram of encoding mechanism based on the different peak intensity ratios; (B) fluorescence emission intensity stability test of UCNMMs excited with a 980 nm NIR laser; (C) fluorescence images of UCNMMs encoded with six distinguishable emission colors UCNs upon a 980 nm optical excitation.

2.3. Immunoassays based on UCNMMs Compared with QDMs. To test whether the immunoassay system based on the UCNMMs can effectively prevent the interference between coded signals and reporter signals, we designed the sandwich immune experiment. There were two different sandwich immune systems, including immunoassay system based on the QDMMs as a control group38 and the immunoassay system based on the UCNMMs as a experimental group. The schematic illustration of immunoassays process is shown in Figure 5A. In the fluorescence spectra of control group, there are two peaks including the luminescence peaks at 650 and 518 nm, which represents the coded signal and reporter signal, respectively. No matter which extra light (UV or Blue- light) was used, both the luminescence peaks at 650 and 518 nm were all existed and overlapped with each other. The phenomenon severely interfered the analysis of reporter signals even at low concentrations, which leads to inaccurate test results. In the experimental group, we found an interesting phenomenon. After being excited with blue light for observing reporter signals from fluorescein isothiocyanate (FITC) labeled rabbit antimouse IgG, only the detection signal located at 520 nm is observed in the fluorescence spectra without any coded signal. Besides, after being excited with NIR light for getting the coded signal from the UCNMMs, only the encoding signal located at 650−680 nm is observed in the fluorescence spectra without any reporter signal. This result means that there is no optical interference during receiving the coded signal and reporter signal. The reason for this is that the UCNMMs and the FITC-labeled rabbit antimouse IgG cannot be excited at the same time using one beam of light source.

In Figure 5B, the intensity of coded signal in the experimental group is much larger than that of reporter signal and these two kinds of signals overlap seriously with each other. As the concentration of mouse IgG decreases, the reporter signal becomes weaker and weaker. At the concentration of 1 μg/mL, the intensity of the reporter signal is nearly close to baseline. The phenomenon severely interferes with analysis of reporter signals, which leads to inaccurate test results. As shown in the Figure 5C, after excited with the blue-light for observing reporter signal from FITC-labeled rabbit antimouse IgG, there is only the detection signal located at 520 nm in the fluorescence spectra without any encoding signal of UCNMMs. With the decrease of the concentration of mouse IgG, the FITC fluorescence signal gradually weakened. Compared with the microspheres encoded with QDs or organic dyes, this is a unique advantage of UCNMMs for appling in the fast and accurate immunoassay. Furthermore, the detection sensitivity is one of the most important performances for immunoassays. To demonstrate the detection sensitivity of UCNMMs, mouse IgG with different concentrations including 1, 5, 10, 40, 60, and 80 μg/mL were set as detection samples. Fluorescence intensities were measured after adding the corresponding FITC-labeled rabbit anti-mouse IgG (Figure 5D). The FITC fluorescence signal is measured on the FL1 channel of the flowcytometry, and in this way we can get high accuracy and low detection limits with a small amount of sample.38 Calculated by the 3-fold standard deviation of the blank samples, the detection limit of mouse IgG is 0.01 ng/mL in our immunoassay. In addition, we have successfully designed double-antibody sandwich immunoassay systems based on the red UCNMMs coated with goat E


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Figure 5. (A) The schematic description, fluorescence images, and spectra of the immunoassays based on UCNMMs (experimental group) and QDMMs (control group); the green arrow points to the free FITC-labeled rabbit antimouse IgG that have not been washed off); (B and C) fluorescence emission spectra of detection signals with different concentrations of Mouse IgG in the immunoassay system based on UCNMMs and QDMMs; (D) standard curve of immunoassay system based on the UCNMMs; the error bar represents standard deviations. oxide (BPO), methacrylic acid (MAA, 98%) and poly(vinyl alcohol) (PVA, Mw 1/4 130 000) were obtained from Fluca. Antibodies such as mouse IgG, goat antimouse IgG, FITC-labeled rabbit antimouse IgG were provided by Beijing Biosciences Co., Ltd. (China). All other chemicals were of analytical grade and obtained from local suppliers. 3.2. Characterization. The morphology of upconverting nanoparticles (UCNs) were observed by Tecnai G2 F20 transmission electron microscope (TEM) operating at an acceleration voltage of 200 kV. The morphology of upconverting nanocrystal encoded microspheres was tested by scanning electron microscopy (SEM, XL30, Philips Corp.). The fluorescence spectra of UCNs and UNencoded microspheres were obtained by luminescence spectrometer with 980 nm laser. True-color fluorescence images were obtained using an inverted Olympus microscope (IX-51) equipped with a broad-band ultraviolet (330−385 nm) light source (100 W mercury lamp), and a 980 nm laser. The magnetization curve of upconverting nanoparticles encoded magnetic microspheres (UN-encoded MM) was tested by vibrating sample magnetometry. The intensiy of encoding and detection signals were measured on a spectral photometer (Carry50). Immunoassays based on UCNMMs were analyzed with flow cytometry (BD FACSAria II). 3.3. Synthesis of UCNs with Different Emission Fluorescences Spectras. UCNs were synthesized by a solvent-thermal process.44,45 Six unique UCNs with different upconversion emission spectras were produced by varying dopant concentrations. NaYF4:Yb,Tm (Y/Yb/Tm = 75/25/0.3) UCNs with blue emission fluorescence were synthesized as follows: YCl3 (0.75 mmol), YbCl3 (0.25 mmol) and TmCl3 (0.003 mmol) were heated to remove water at 110 °C with magnetic stirring for 20 min, until becoming white solid in the four-necked flask. Then, the product was cooled slightly to 80

antimouse IgG and green UCNMMs coated with mouse antirabbit IgG for detecting Mouse IgG and Rabbit IgG simultaneously (Figure 6A), and used FITC-labeled rabbit antimouse IgG and phycoerythrin (PE)-labeled goat antiRabbit IgG to be reporter signals correspondingly. It can be seen from Figure 6B that each sandwich immunoassay system works well for double detection. FITC/PE-labeled antibodies were given emission at 520 and 578 nm respectively under blue light excitation (Figures S12 and S14). In the flow cytometric analysis of double detection (Figure 6C), FITC/PE-labeled antibodies in one sample can be detected on FL1, FL2 channel separately after fluorescence compensation (Q1, PE positive, FITC negative; Q2, PE positive, FITC positive; Q3, PE negative, FITC positive; Q4, PE negative, FITC negative; M1, postive; M2, negative).

3. MATERIALS AND METHODS 3.1. Materials. Yttrium(III) chloride hexahydrate (YCl3·6H2O, 99.99%), ytterbium(III) chloride hexahydrate (YbCl3·6H2O, 99.99%), erbium(III) chloride hexahydrate (ErCl3·6H2O, 99.99%), manganese(II) chloride tetrahydrate (MnCl2·4H2O), thulium(III) chloride hexahydrate (TmCl3.6H2O), oleic acid (OA,90%), 1-octadecene (ODE,90%), ammonium fluoride (NH4F, 99.99%), styrene (St, 99%), poly(vinylpyrrolidone) (PVP K-40), ethylene glycol dimethacrylate (EGDMA,98%), sodium dodecyl sulfate (SDS), iron(III) acetylacetonate (Fe(C5H7O2)3), oleylamine (OLA), 1,2-dihydroxydodecane and Dibenzyl Ether were purchased from Sigma-Aldrich. 2,2′Azobis(2-methylpropionamide)dihydrochloride (AIBA), benzoyl perF


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Figure 6. (A) Schematic illustration of double-antigen sandwich immunoassay based on the UCNMMS with orange/green emission color; (B) fluorescence images of the double-antibody sandwich immunoassay; (C) flow cytometry analysis of double- antibody sandwich immunoassay. °C and added 6 mL OA and 15 mL ODE. The solution was heated to 160 °C to form a homogeneous solution, and then cooled to 60 °C. Then, 5 mL of methanol solution containing NaOH (2.5 mmol) and NH4F (4 mmol) was added into the flask and stirred for 20 min. Subsequently, the solution was heated to 80 °C to remove methanol and degassed at 100 °C three times during 30 min. Afterward, the temperature was increased to 300 °C and kept for 1 h under argon protection. Nanocrystals were precipitated from the solution with ethanol (8000 rpm, 10 min), and the redundant salt was washed with ethanol/cyclohexane (1:1 v/v, 2000 rpm, 5 min) three times, and finally, the nanocrystals were resolved in 20 mL chloroform. NaYF4:Yb,Tm,Er (Y/Yb/Tm/Er = 75/25/0.3/0.2) nanocrystals with cyan fluorescence, NaYF4:Yb,Er (Y/Yb/Er = 80/20/2) nanocrystals with green fluorescence and NaYF4:Yb,Er (Y/Yb/Er = 80/20/0.4) nanocrystals with yellow fluorescence were synthesized as the method above. Furthermore,the synthesis process of NaYF4:Yb,Tm@ 0.6(NaYF4:Yb,Er) nanocrystals with sky-blue fluorescence and NaYF4:Yb,Er@Mn (Y/Yb/Er/Mn = 35/20/1/45) nanocrystals with orange fluorescence can be found in the Supporting Informations. 3.4. Preparation of UCNMMs. The highly cross-linked monodisperse porous poly(styrene-co-EGDMA-co-MAA) beads (PSEMBs) and magnetic nanoparticles were prepared according to

our Supporting Informations. The self-healing encapsulation method was carried out for preparation of UCNMMs. First, MNs were dispersed in chloroform (55 μL) and PSEMBs were dispersed in 3−5 mL chloroform/isopropanol(95:5,V/V). The mixture was fully dispersed by ultrasonic vibration for 20 min, and evaporated in the vacuum drying oven at 30 °C for 12 h. The magntic microspheres were obtained by centrifugation and wash for several times with ethanol/cyclohexane (1:1, v/v); After that, magntic microsphere were dispersed in ODE (5 mL), and UCNs were dispersed in chloroform (750 μL). The mixture was fully dispersed by ultrasonic vibration, added into a flask and stirred at 50 °C for 1 h. Then the temperature was slowly raised to 180 °C and kept warm for 30 min. Finally cooled rapidly to room temperature. The argon protection ran through the entire process. UCNMMs were centrifugated and washed with ethanol/cyclohexane (1:1, v/v) for several times. 3.5. UCNMMs based sandwich immunoassays. First, use EDC and NHS as activators in 500 μL PBS solution (0.01M,pH 7.4) to rotatable culture for 30 min with UCNMMs. The UCNMMs were then washed with PBS three times. The activated UCNMMs were coated with goat Antimouse IgG by to incubate for 2 h, subsequently stored in PBS buffer solution (0.01 M, pH 7.4, 2% BSA) overnight at 4 °C. The molar ratio of UCNMMs/antibody/EDC/NHS was 1/3/3. Then, different concentrations gradient of mouse IgG to be tested and G


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FITC-labeled rabbit antimouse IgG for detecting were added in order to incubate for 1h and then washed washed by PBST (0.05%T-20) twice. Throughout the whole process, UCNMMs Conjugated antibodies were captured and separated quickly by the magnet adsorption within 2 min.46,47 For multiple detection, the prepared UCNMMs conjugated goat antimouse IgG or mouse antirabbit IgG were mixed, then added the mixture including mouse IgG and rabbit IgG. After incubating for 1 h, UCNMMs were washed with PBST twice. Last, we added the mixture of FITC−rabbit antimouse IgG and PE-labeled goat anti-Rabbit IgG, incubated the mixture for 30 min, and washed the mixture with PBST twice.

4. CONCLUSION In summary, the results demonstrate that UCNMMs exhibit good sphericity and uniform particle size distribution. They can be easily assembled and labeled with biomolecules and possess a large optical encoding bandwidth capability. Compared with the immunoassay based on the downconversion target materials (organic dyes or quantum dots), there is no optical interrelationship between the upconversion encoding signal and any reporter dyes during the immunoassays. So, the detection labels can be selected in a wide emission range. UCNMMs fabricated by the self-healing encapsulation methods have excellent binding capacity for biomolecules and are suitable for fast separation, which has large potential applications in multiplexed immunoassays.

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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/acsami.5b09913. TEM images of particle sizes, diameter distributions of UCNs, X-ray diffraction images of UCNs, SEM images of synthetic polystyrene microspheres, the conductometric titration of microspheres, thermal-gravimetric curve of UCNMMs, schematic diagram and microscope photos of double-antibody sandwich immunoassay. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions ∥

Ying Zhang and Chunhong Dong contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the Natural Science Foundation of China (51373117, 51303126, and 3140101465), National High Technology Program of China (2012AA022603), Tianjin Natural Science Foundation (13JCZDJC33200 and 15JCQNJC03100), Doctoral Base Foundation of Educational Ministry of China (20120032110027).

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REFERENCES

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