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Upconversion Nanocrystals Mediated LateralFlow Nanoplatform for In Vitro Detection Zhiqin Liang, Xiaochen Wang, Wei Zhu, Pingping Zhang, Yongxin Yang, Chongyun Sun, Junjie Zhang, Xinrui Wang, Zheng Xu, Yong Zhao, Ruifu Yang, Suling Zhao, and Lei Zhou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14906 • Publication Date (Web): 09 Jan 2017 Downloaded from http://pubs.acs.org on January 10, 2017

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ACS Applied Materials & Interfaces

Upconversion Nanocrystals Mediated Lateral-Flow Nanoplatform for In Vitro Detection Zhiqin Liang1,$, Xiaochen Wang2,3,4,$, Wei Zhu1, Pingping Zhang2,3, Yongxin Yang1, Chongyun Sun2,3,5, Junjie Zhang1, Xinrui Wang2,3,6, Zheng Xu1, Yong Zhao2,3, Ruifu Yang2,3, Suling Zhao1*, and Lei Zhou2,3* 1

Institute of Optoelectronics Technology, Key Laboratory of Luminescence and Optical

Information, Ministry of Education, Beijing Jiaotong University, Beijing 100044, P. R. China 2

Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity,

Beijing Institute of Microbiology and Epidemiology, Beijing 100071, P. R. China 3

Beijing Key Laboratory of POCT for Bioemergency and Clinic (No. BZ0329), Beijing 100071,

P. R. China 4

College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, P.

R. China 5

Department of Clinical Laboratory, Chinese People’s Liberation Army General Hospital, Beijing

100853, P. R. China 6

Institute for Plague Prevention and Control of Hebei Province, Zhangjiakou 75000, P. R. China

$ Z.L. and X.W. contributed equally to this work.

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ABSTRACT Upconversion phosphors (UCPs) that are free from interference from biological sample autofluorescence have attracted attention for in vivo and in vitro bio-applications. However, UCPs need to be water-dispersible, nanosized, and highly luminous to realize broad applications. Therefore, the aim of this research is to develop UCPs that meet these comprehensive criteria for in vitro diagnosis. To combine nanosize with high luminous intensity, β-NaYF4:Yb3+,Er3+ upconversion nanocrystals (UCNPs) codoped with Li+ and K+ are prepared that display high upconversion intensities as well as small size. The strongest green and red emissions of the Na0.9Li0.07K0.03YF4:Yb3+,Er3+ nanocrystals are increased by 7 and 10 times, respectively, compared with those of the undoped NaYF4:Yb3+,Er3+ nanocrystals. A mild sol–gel surface modification method is used to produce water-phase dispersions and allow covalent biomolecule conjugation. The bioactivated UCNPs are used as a bioreporter and integrated with a classical lateral flow assay to establish an assay to accomplish simultaneous dual-target detection of Yersinia pestis and Burkholderia pseudomallei. The assay achieves a sensitivity of 103 CFU/test without cross-interference between two targets. The research provides a way to produce UCNPs with comprehensive properties for use as excellent optical reporters in in vivo and in vitro bio-applications. KEYWORDS: NaYF4:Yb,Er nanocrystals, upconversion fluorescence, Li+ and K+ codoping, covalent biomolecule conjugation, dual-target detection


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1. INTRODUCTION In recent years, near-infrared (NIR) upconversion phosphors (UCPs) have attracted great attention because of their remarkable potential in bio-applications.1-5 In NIR UCPs, the upconversion (UC) process involves converting two or more low-energy photons to a highenergy output photon under NIR excitation.6 Based on this unique emission process, UCP particles own many advantages over traditional downconversion nanoparticles, such as high physical and chemical stability, minimal autofluorescence background, and low cytotoxicity, which make them suitable for bio-applications as optical reporters.7-12 Present bio-applicationoriented research on UCP particles can be divided into that considering nanoparticles smaller than 50 nm (called upconversion nanoparticles [UCNPs]), which are attractive for in vivo bioapplications such as in vivo imaging13-15 and targeted therapy,16-18 and that focusing on upconversion submicrometer-sized particles (UCSMPs). Usually, UCSMPs have a diameter of

around

400

nm

and

are

promising

for

in

vitro

applications

including

immunohistochemistry,19 biochips,20 homogeneous energy transfer,21 and lateral flow (i.e., immunechromatography)22-27 for point-of-care testing (POCT); our team is also working on this topic.28-32 To realize bio-applications of UCP particles, they should meet three criteria regarding surface properties (hydrophilic or hydrophobic), size, and luminous intensity. In particular, the surface properties of UCP particles are critical; they need to fit for the water phase for bioapplication. Accordingly, previously reported UCP particles for both in vivo and in vitro applications all possess hydrophilic surfaces.33, 34 However, it is difficult for the other two properties to coexist because of the inverse relation between the size and luminous intensity of UCNPs, which results in inevitable defects in UCP particles being designed for in vivo and in vitro uses. For in vivo applications, particles must possess a small diameter to penetrate into tissue and transfer through circulation, leading to the sacrifice of luminous intensity. 3 ACS Paragon Plus Environment


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Moreover, hydrophilic treatment (such as using potassium permanganate to modify oleic acid) after synthesis in the oil phase usually produces defects on the particle surface, which makes the luminous intensity decrease further. Regarding in vitro applications, to obtain higher luminous intensity, the particles usually have a large diameter (usually ~400 nm), which leads to

considerable

steric

hindrance,

especially

in

microporous

material-based

immunochromatography. Therefore, combining the advantages of the two types of UCP particles to develop a new kind of particles with the properties of water-phase dispersion, nanoscale size, and high luminous intensity will greatly promote their application in biology. According to these comprehensive requirements for bio-application of UCNPs, here we focus on systematically researching synthesis and surface treatment parameters in an attempt to balance the tradeoff between the size and luminous intensity of UCP particles and minimize the damage to luminous efficiency caused by hydrophilic treatment, respectively. Firstly, based on a series of experiments involving codoping with Li+ and K+ to enhance UC intensity and control particle morphology, we prepare oil-phase Na0.9LixK0.1−xYF4:Yb,Er UCNPs with diameters of 25-40 nm and high luminous intensity, in which the concentration of Yb3+ and Er3+ are 20% and 2%, respectively. Then, hydrophilic surfaces are obtained using a mild, general sol–gel method while maintaining both the nanoscale size and high luminous intensity of the UCNPs. Finally, by combining UCNPs as the optical reporter with a lateral flow assay, we develop an UCNP-based lateral flow (UCNP-LF) assay and achieve the dual detection of large targets using Yersinia pestis and Burkholderia pseudomallei as the model (Figure 1). Such detection has not been achieved previously because of fatal steric hindrance resulting from the specific combination of UCSMPs (~400 nm) with bacteria cells (~2 µm). In our previous research,35 we reported several examples of 50 nm UCNPs combined with lateral flow assays for in vitro bio-applications, but did not explore the UCNPs thoroughly; therefore, UCNPs are the focus of this paper. 4 ACS Paragon Plus Environment

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Figure 1. Schematic diagram of the structure and detection results of the upconversion nanoparticle-based lateral flow (UCNP-LF) assay. (a) UCNP-LF strip with a similar structure to that of a traditional gold colloid -based lateral-flow strip. The sample pad, conjugate pad, analytical membrane, and absorbent pad were mounted on a laminated card with proper overlaps. All bio-molecules for specific detection were fixed in advance, including the 5 ACS Paragon Plus Environment


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UCNP–antibody conjugate in the conjugate pad, and antibodies for test and control lines on the analytical membrane. For (b) B. pseudomallei or (c) Y. pestis detection, a pair of specific antibodies for the target were used to conjugate with UCNPs and as the test line, respectively. During positive detection, the captured target formed a UCNP–antibody–target–antibody complex on the test line and then produced a detectable, quantifiable optical signal that depended on the relationship between the ratio of the peak areas of the test and control lines (Vt/Vc) and the target concentration in the sample. (d) For dual-target detection of B.

pseudomallei and Y. pestis, two test lines were included with each corresponding to a certain target, allowing two targets to be qualitatively and quantitatively detected during a single operation.

2. RESULTS AND DISCUSSION 2.1 Structure and Morphology of Na0.9LixK0.1−xYF4:Yb, Er samples The XRD patterns and the main diffraction peak of Na0.9LixK0.1−xYF4:Yb, Er UCNPs with different x values of 0, 0.03, 0.05, 0.07, and 0.1 are shown in Figure 2a. When x was ≤0.1, all the diffraction peaks could be indexed as pure β-NaYF4 (JCPDS No.16-0334) (Figure 2a(1)), indicating that low contents of Li+ and K+ codoping do not influence the crystalline phase of NaYF4. In the hexagonal NaYF4 lattice, Na+ will be replaced by Li+ and K+ in the codoped structure because these ions have the same valence. When a low content of Li+ is doped into the NaYF4 lattice, Li+ may either substitute the sites of Na+ or occupy interstitial sites (Figure 3a and b), because Na+ has a bigger ionic radius (0.97 Å) than that of Li+ (0.68 Å).36 Conversely, the ionic radius of K+ (1.38 Å) is larger than that of Na+,37 so K+ cannot occupy interstitial sites because it will need to cost a lot of energies and result in unstable crystal structure. Thus, K+ only substitutes Na+ sites (Figure 3c). 6 ACS Paragon Plus Environment

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Combining the above analysis with our experimental XRD patterns in Figure 2a(2), it is obvious that the main diffraction peak moves towards larger angles as x increases to 0.07, and then shifts to smaller angles as x increases further to 0.1. According to Bragg’s law

2dsinθ=nλ, where d is the interplanar distance, θ is the diffraction angle, and λ is the diffraction wavelength, d decreases and θ increases. As x gradually increases from 0 to 0.07, some Na+ may be substituted by both Li+ and K+, resulting in d decreasing and θ increasing. With further increasing x to 0.1, more Li+ may occupy the interstitial sites, so d increases and

θ decreases accordingly. It is worth noting that the angles of the main diffraction peaks of the products with ≤ 0.05 are all smaller than those of Li+ and K+-free NaYF4:Yb, Er nanocrystals. This means that doping with more K+ ions causes the crystal lattice to expand. On the contrary, θ becomes larger when x exceeds 0.05, which indicates that codoping with more Li+ causes the crystal lattice to contract. Therefore, changes of the surrounding environment of Er3+ and uniform crystal size may be realized by codoping with Li+ and K+. In addition, the contracted crystal lattice should facilitate the interaction between Er3+ and Yb3+ and strengthen the intensity of UC emission. Transmission electron microscopy (TEM) images indicate that all nanocrystals are uniform in size and morphology, as illustrated in Figure 2b. The nanocrystals are spherical, and their morphology is not affected by introduction of foreign ions. The average nanoparticle sizes are 26, 33, 29, 43, 40, and 26 nm for NaYF4:Yb, Er nanocrystals and Na0.9LixK0.1−xYF4:Yb,Er UCNPs with x values of 0, 0.03, 0.05, 0.07, and 0.1, respectively. Particle size differs slightly with the contents of Li+ and K+, but is still similar enough to that of undoped NaYF4:Yb,Er to fall within the useful size range for in vitro biological applications.



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Figure

2.

Characterization

of

UCNPs.

(a)

X-ray

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diffraction

patterns

of

Na0.9LixK0.1−xYF4:Yb,Er nanocrystals and standard data for hexagonal NaYF4 (JCPDS No. 16-0334). The scanning angle range was 10°–80° for (1), and 42.5°–45.5° for (2). (b) TEM images of (1) NaYF4:Yb,Er nanocrystals and Na0.9LixK0.1−xYF4:Yb,Er nanocrystals with (2) x=0, (3) x=0.03, (4) x=0.05, (5) x=0.07, and (6) x=0.1. Scale bar is 100 nm. (c) Upconversion emission spectra of the Na0.9LixK0.1−xYF4:Yb,Er nanocrystals with different x values. The inset shows the integral intensity of green and red emission as a function of x. (d) Digital photographs

of

upconversion

emission

of

(1)

NaYF4:Yb,Er

nanocrystals

and

Na0.9LixK0.1−xYF4:Yb,Er nanocrystals with (2) x=0, (3) x=0.03, (4) x=0.05, (5) x=0.07, and (6) x=0.1 under the excitation of a 980 nm laser diode.


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Figure 3. Changes of the NaYF4 crystal lattice upon doping with foreign ions: (a) substitution by small ions, (b) interstitial occupation by small ions, and (c) substitution by large ions. Red circles represent dopant ions.

2.2 Upconversion Fluorescence of Na0.9LixK0.1−xYF4:Yb, Er Samples The UC spectra of the samples were recorded under identical conditions. As illustrated in Figure 2c, the dominant green emissions at 525 and 541 nm are assigned to the 2H11/2→4I15/2 and 4S3/2→4I15/2 transitions, respectively. The red emission at 655 nm is ascribed to the 2

F9/2→4I15/2 transition. Pump power dependence of the green and red emissions of the samples

have been present as supplementary Figure S1. . The intensity of these emission peaks generally increased upon introducing Li+ and K+ compared with that of the emission peaks of undoped nanoparticles. The maximum UC emission was observed when x was 0.07, with the intensities of green and red emissions enhanced by 7 and 10 times, respectively, compared with those of the emissions of the undoped sample. The inset in Figure 2c is the integral intensity of green and red emission as a function of x, which clearly shows similar variation trends for both emission signals. We note that the intensities of green and red UC emissions are both enhanced as x increases from 0 to 0.07, and then weaken as x further increases to 0.1.



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The initial increase of the UC intensity with rising x may be associated with lowered crystal field symmetry upon doping with foreign ions and the enlarged size of the nanoparticles after doping. It is well known that UC emission depends on the intra 4f transition probabilities of rare-earth ions, which is influenced by their local crystal field symmetry. Lower crystal field symmetry may lead to more thorough suppression of the 4f electric dipole transition and therefore result in greater luminescence intensity.38, 39 When Li+ and K+ are introduced into the NaYF4 lattice, local crystal field symmetry may decrease and thus enhance the UC emission intensity. When x is 0.07, the maximum intensities of both green and red emissions are observed. In this sample, in addition to the asymmetric environment, the contracting crystal lattice results in a stronger interaction between Er3+ and Yb3+. Besides, the slightly enlarged particle size may also contribute to the enhanced UC emission. The subsequent decrease in UC emission intensity as x increases above 0.7 is attributed to the increasing symmetry of the environment around Er3+ compared with that when x is less than 0.07. Of course, the smaller particle size is also another factor for the decreased intensity of UC emission. However, the emission intensity is still greater than that of undoped NaYF4:Yb,Er nanoparticles because the symmetry of samples with x >0.07 is still lower than that of the undoped nanoparticles. The digital photographs in Figure 2d present an intuitive insight into the UC emission of the samples. The changes in UC emission intensity can be clearly observed by the naked eye; fluorescence intensity is markedly increased by introduction of foreign ions. Moreover, the visually observed change of fluorescence intensity is consistent with the trend in the inset of Figure 2c.

2.3 Surface Modification of UCNPs The optimized Na0.9Li0.07K0.03YF4:Yb,Er nanoparticles with a relatively small size (~40 nm) 10 ACS Paragon Plus Environment

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and the highest UC emission intensity of the samples are monodisperse in oil phase. Following amino and aldehyde modification and antibody conjugation, these nanoparticles maintained their monodisperse character in the aqueous phase (Figure 4). Consequently, different from strong oxidizer-based surface modification that sacrifices UC intensity to obtain a hydrophilic surface, the sol–gel method used here was able to combine a hydrophilic surface, small size, and high luminous intensity together, setting a solid foundation to replace UCSMPs commonly used in vitro.

Figure 4. TEM images of (a) amino-modified Na0.9Li0.07K0.03YF4:Yb,Er UCNPs, (b) aldehyde-modified UCNPs, and (c) UCNP–antibody conjugates in the aqueous phase. Scale bar is 200 nm.

2.4 In Vitro Bio-application of UCNPs 2.4.1 Sensitivity and Quantitative Capability of the UCNP-LF Assay Series of Y. pestis and B. pseudomallei solutions with different concentrations obtained by dilution with phosphate buffer were detected by Ype-UPT-LF and Bps-UPT-LF strips, respectively, and then they were both detected simultaneously by a YB-UPT-LF strip (the 11 ACS Paragon Plus Environment


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original scanning figure of the up-converting phosphor technology-based biosensor (UPT biosensor) is provided as supplementary Figure S2). The sensitivities of Ype-UPT-LF and Bps-UPT-LF were 102 and 103 CFU/test, respectively. The sensitivity of YB-UCNP-LF for detection of both Y. pestis and B. pseudomallei was 103 CFU/test, which is ten-fold lower than that of Ype-UCNP-LF and equal to that of Bps-UCNP-LF. A standard curve with the logarithm of the ratio of the peak areas of the test and control lines (Vt/Vc) minus cutoff as the x-axis against the logarithm of the amount of bacteria per test is plotted in Figure 5a. The quantitative ranges for Ype-UPT-LF and Bps-UPT-LF were from 102 to 105 and 103 to 106 CFU/test, respectively, and the quantitative range of YB-UPTLF for both Y. pestis and B. pseudomallei was from 103 to 105 CFU/test. The correlation coefficients of linear regression analysis were all above 0.9, demonstrating that the accuracy of the UCNP-LF assay was very high (Figure 5a). 2.4.2 Simultaneous Detection by UCNP-LF Assay Because we systematically evaluated the specificity of antibodies for Y. pestis and B. pseudomallei in our previous research,40,

41

the two most important properties of multiple

detection are sensitivity and anti-interference in the presence of the other detected target at high concentration. Therefore, series of Y. pestis or B. pseudomallei solutions with the other species as the diluent (108 CFU/mL) were detected by YB-UCNP-LF. Here, Vt1/Vc represents the detection result for Y. pestis, and Vt2/Vc that for B. pseudomallei. With 108 CFU/mL of non-target bacteria as interference, the sensitivity of YB-UCNP-LF for Y. pestis and B. pseudomallei remained equal at 103 CFU/test without false positives for high-interference negative samples (i.e., when the concentration of target bacteria was 0 CFU/mL and that of non-target bacteria was 108 CFU/mL). Thus, YB-UCNP-LF showed excellent antiinterference behavior (Figure 5b and c). 12 ACS Paragon Plus Environment

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The performance of the dual-target UCNP-LF compared with that of the single-target ones is poorer in terms of both sensitivity (during Y. pestis detection, the sensitivity is 102 CFU/test for Ype-UPT-LF and 103 CFU/test for YB-UPT-LF) and quantitative range (102–105 and 103– 105 CFU/test for Y. pestis detected by Ype-UPT-LF and YB-UPT-LF, respectively, and 103– 106 and 103–105 CFU/test for B. pseudomallei detected by Bps-UPT-LF and YB-UPT-LF, respectively). This results from the steric hindrance of the large bacteria cells (about ~2 µm) being greater when two targets are detected simultaneously. However, the nanoscale size of the UCNPs made dual-target detection possible because of their excellent anti-interference, which is usually limited by the marked steric hindrance of UCSMPs.

Figure 5. UCNP-LF assays using UCNPs (Na0.9Li0.07K0.03YF4:Yb,Er) as the bio-reporter. (a) Standard curves of Ype-UPT-LF, Bps-UPT-LF, and YB-UPT-LF showing the sensitivity, quantitative range, and accuracy of the UCNP-LF assays. Simultaneous detection results for (b) Y. pestis and (c) B. pseudomallei by the YB-UCNP-LF assay reveal that YB-UCNP-LF



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retains the same performance for each target with or without the interference of 108 CFU/mL non-target bacteria, resulting in excellent anti-interference.

3. CONCLUSION Considering the comprehensive criteria of UCP for in vivo and in vitro applications, including water phase dispersion, nanoscale size, and high luminous intensity, we prepared βNaYF4:Yb3+,Er3+ UCNPs codoped with different contents of Li+ and K+ to enhance UC emission intensity without changing morphology and size. The strongest green and red emission intensities were obtained for the nanocrystals codoped with 7 mol% of Li+ and 3 mol% of K+, which were increased by 7 and 10 times compared with those of the undoped NaYF4:Yb,Er sample, respectively. Then, a mild sol–gel method was used to form water phase dispersions to use the bioactivated UNCPs as optical reporters. Classical lateral flow assays were used to verify the comprehensive properties of the bio-activated UCNPs. A dualtarget YB-UCNP-LF assay for qualitative and quantitative detection of large targets (including Y. pestis and B. pseudomallei) was established by overcoming the low luminous intensity or large scale of previous UCP materials. A sensitivity of 103 CFU/test and excellent anti-interference between two detected targets were achieved. The optimized UCNPs provide a reference for the subsequent research of UCP materials for bio-applications and an alternative optical reporter for biotechnology innovation.

4. EXPERIMENT SECTION 4.1 Reagents and Instruments Yttrium(III) chloride (YCl3, 99%), ytterbium(III) chloride (YbCl3, 99%), erbium(III) chloride (ErCl3, 99%), oleic acid (OA, 90%), 1-octadecene (ODE, 90%), ammonium fluoride (NH4F, 14 ACS Paragon Plus Environment

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98%), sodium hydroxide (NaOH, 99.99%), absolute methanol (C2H6O, 90%), cyclohexane (C6H12, 90%), triethoxyaminopropylsilane (APES, 99.99%), tetraethylorthosilicate (TEOS, 98%), albumin bovine V from bovine serum albumin (BSA, 98%), and casein were all purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade, used without further purification, and supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) unless otherwise specified. Nitrocellulose membranes (SHF 1350225) and glass fibers (GFCP20300) were obtained from Millipore (Bedford, MA, USA). Papers (Nos. 470 and 903) were purchased from Schleicher & Schuell, Inc. (Keene, NH, USA). Plastic cartridges and laminating cards were designed by our group and manufactured by Shenzhen Jincanhua Industry Co. (Shenzhen, China) and Shanghai Liangxin Biotechnology Co. (Shanghai, China), respectively. Monoclonal antibodies (McAb) against Y. pestis or B. pseudomallei, goat anti-mouse IgG, goat IgG and rabbit anti-goat IgG were prepared by our group. The pure cultures of Y. pestis and B. pseudomallei were preserved in our laboratory and used under appropriate bio-safety measures. The instruments used included a supercentrifuge (5417R, Eppendorf, Germany), thermomixer compact (Eppendorf, Germany), IsoFlowTM dispenser (Imagene Echnology, United States), protein nucleic acid spectrophotometer (DU640, Beckman Coulter, Canada), numerical control high-speed interrupted cutting machine (ZQ4000, Shanghai Goldbio Tech Co, China), X-ray diffractometer (D8 focus, Bruker, Germany), transmission electron microscope (JEM-1400, JEOL Ltd, Japan), fluorescence spectrometer system (ZOLIX, Beijing ZOLIX Instruments Co., China). The UPT biosensor was designed and fabricated by our group and Shanghai Institute of Optics and Fine Mechanics, Chinese Academy of Sciences (Shanghai, China). 4.2 Synthesis of NaYF4: Yb, Er and Na0.9LixK0.1−xYF4: Yb, Er Nanocrystals



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In a typical synthesis of NaYF4:20% Yb, 2% Er, YCl3 (0.78 mmol), YbCl3 (0.2 mmol), and ErCl3 (0.02 mmol) were added to a 50-mL flask containing OA (6 mL) and ODE (15 mL). The reaction mixture was heated to 160 °C for 30 min under vigorous magnetic stirring and then cooled down to room temperature. Afterwards, methanol solution (10 mL) containing NaOH (2.5 mmol) and NH4F (4 mmol) was added, and the resulting mixture was stirred at 70 °C for 30 min to remove methanol. Subsequently, the solution was heated at 300 °C under argon atmosphere for 90 min. After cooling to room temperature, the obtained nanocrystals were precipitated and washed with ethanol several times, and finally dried at 60 °C for 12 h. The other products were synthesized using LiOH and KOH instead of NaOH while keeping the other conditions the same. The prepared nanocrystals were identified by powder XRD using a Bruker D8 focus diffractometer with Cu-Kα radiation (λ=1.540 Å). TEM measurements were carried out on a JEM-1400 transmission electron microscope operating at an acceleration voltage of 100 kV. UC emission spectra were recorded on a ZOLIX fluorescence spectrometer equipped with an external 980-nm diode laser. All measurements were performed at room temperature. 4.3 UCNP Surface Modification and Bio-conjugation The optimized Na0.9LixK1−xYF4:Yb,Er nanoparticles were conjugated with the antibodies against Y. pestis and B. pseudomallei. The modification method could be divided into two steps: amino and aldehyde modification. To conjugate amino groups onto the UCNPs, UCNPs (100 mg) were dissolved in isopropanol (50 mL). TEOS (5 µL) was added to the mixture, which was then stirred vigorously at 42 °C for 2 h. The UCNPs was washed twice with isopropanol and resuspended in isopropanol (50 mL). To the mixture was added APTES (10 µL), and then it was magnetically stirred for 3 h before the solvent was totally evaporated to dryness. The UCNPs were washed with isopropanol and then incubated at 110 °C for 6 h. The 16 ACS Paragon Plus Environment

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resulting amino-UCNPs were resuspended in 2% glutaraldehyde solution diluted with phosphate buffer (pH=7.2, 0.03 mol/L) and magnetically stirred for 3 h. The produced aldehyde-UCNPs were collected by centrifugation and washed three times with phosphate buffer (pH=7.2, 0.03 mol/L). Aldehyde-UCNPs (500 µg) were added to phosphate buffer (500 µL, pH=7.2, 0.03 mol/L) along with antibody (5 µg). The mixture was shaken for 1 h. BSA (50 µg) was added to the solution, which was shaken for another 1 h. The resulting UCNP–antibody conjugate was collected by centrifugation and dispersed in conjugate buffer (500 µL, phosphate buffer [pH=7.2, 0.03 mol/L], containing 1% BSA, 0.1% NP40, 1% trehalose) for future use. 4.4 UCNP-LF Strip Development and Detection 4.4.1 Single-Target and Dual-Target Strip Development UCNP-LF strips for single-target detection of Y. pestis or B. pseudomallei were first produced by imitating the construction of traditional gold colloid-based lateral flow strips (Figure 1a), and named Ype-UCNP-LF and Bps-UCNP-LF, respectively. The strips were fabricated as follows. First, UCNP–antibody conjugate solution (1 mg/mL) was added to the glass fiber and then rapidly dried at 45 °C for 30 min for use as a conjugate pad. McAb against Y. pestis or McAb against B. Pseudomallei (2 mg/mL) and goat anti-mouse IgG (1 µL/cm) were dispensed by an IsoFlowTM dispenser on a nitrocellulose membrane as a test line (T) and control line (C), respectively, and dried at 37 °C for 30 min (see Figure 1b and c). To assemble LF strips, the conjugate pad, nitrocellulose membrane, sample pad, and absorbent pad were mounted on a laminated card with suitable overlaps, and then cut to a width of 4 mm to install in a plastic shell.



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Dual-target UCNP-LF strips for simultaneous detection of Y. pestis and B. pseudomallei were also fabricated in a similar manner, and denoted YB-UCNP-LF. To prepare this conjugate pad, UCNP–McAb against Y. pestis, UCNP–McAb against B. pseudomallei, and UCNP–goat IgG conjugates were mixed together with concentrations of 1 mg/mL. McAb against Y. pestis, McAb against B. pseudomallei, and rabbit anti-goat IgG with concentrations of 2 mg/mL were dispensed on a nitrocellulose membrane as test line 1 (T1), test line 2 (T2), and a C line, respectively (Figure 1d). 4.4.2 Detection and Result Analysis A sample (10 µL) was mixed with sample-treating buffer (90 µL, phosphate buffer [pH=7.2, 0.03 mol/L] containing 2% BSA, 0.5% NaCl, 0.1% TritonX-100, and 0.05% SDS). The solution was added dropwise onto a strip, which was analyzed by the UPT biosensor after standing 15 min to allow sample flow and immunoreaction. In positive detection, the detected target first combines with a UCNP–antibody conjugate located in the conjugate pad, and then is captured by another pairing antibody immobilized as the T line on the analytical membrane during lateral flow. This immobilizes the UCNPs with the detected target as a bridge to give a detectable, quantifiable signal corresponding to the target concentration in the sample. In negative detection, because of the absence of the target, UCNPs are only captured by the C line, so the optical signal of UCNPs is detected on the C line but not the T line. The peak areas corresponding to the T and C lines are denoted as Vt and Vc, respectively, and the ratio of Vt to Vc (Vt/Vc) is used to judge the concentration of the target in the tested sample qualitatively and quantitatively. By including two T lines, dual targets could be detected simultaneously (Figure 1). 4.5 Performance Evaluation of the UCNP-LF Assay 4.5.1 Sensitivity and Quantitative Capability Evaluation 18 ACS Paragon Plus Environment

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The pure culture of Y. pestis or B. pseudomallei was serially diluted ten-fold with phosphate buffer to give concentrations from 104 to 108 CFU/mL and then detected by Ype-UCNP-LF or Bps-UCNP-LF, respectively, or both were detected using YB-UCNP-LF. The cutoff threshold was determined using phosphate buffer as the negative sample, and equaled the mean of Vt/Vc plus three times the standard deviation (MeanVt/Vc + 3SD). Samples with Vt/Vc larger than cutoff threshold were positive and vice versa. The lowest concentration that gave Vt/Vc larger than the cutoff threshold was determined as the limit of detection, which represents the sensitivity of the assay. 4.5.2 Simultaneous Detection by a Dual-Target UCNP-LF Strip The pure culture of Y. pestis was diluted to 0, 104, 105, 106, 107 and 108 CFU/mL using phosphate buffer and 108 CFU/mL B. pseudomallei as the diluent, respectively. For the standard samples of B. pseudomallei with concentrations of 0, 104, 105, 106, 107 and 108 CFU/mL, the diluent was phosphate buffer and 108 CFU/mL Y. pestis respectively. All samples were tested using YB-UCNP-LF three times. Under non-target interference, if the Vt/Vc ratio of the negative sample was still lower than the cutoff value, it means that the dualtarget

UCNP-LF

assay

shows

good

anti-cross-interference

simultaneously detected targets.


performance for two


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ASSOCIATED CONTENT Supporting Information. The original scanning figure of Ype-UPT-LF, Bps-UPT-LF and YB-UPT-LF by UPT biosensor. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Email address: [email protected]; [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We thank the National High Technology Research and Development Program of China (863 Program) under Grant No. 2013AA032205, the National Natural Science Foundation of China under Grant No. 51272022 and Beijing Nova Program of China (Grant No. Z151100000315086).


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TOC


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