Metal (Au, Pt) NanoparticleâLatex Nanocomposites as Probes for

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Biological and Medical Applications of Materials and Interfaces

Metal (Au, Pt) Nanoparticle–Latex Nanocomposites as Probes for Immunochromatographic Test Strips with Enhanced Sensitivity Yasufumi Matsumura, Yasushi Enomoto, Mari Takahashi, and Shinya Maenosono ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b11745 • Publication Date (Web): 05 Sep 2018 Downloaded from http://pubs.acs.org on September 6, 2018

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ACS Applied Materials & Interfaces

Metal (Au, Pt) Nanoparticle–Latex Nanocomposites as Probes for Immunochromatographic Test Strips with Enhanced Sensitivity Yasufumi Matsumura,1* Yasushi Enomoto,1 Mari Takahashi,2 Shinya Maenosono2*

1

New Materials Development Center, Research & Development Division, Nippon Steel & Sumikin Chemical Co., Ltd., 1-Tsukiji, Kisarazu, Chiba 292-0835, Japan

2

School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan

Corresponding Authors *E-mail: [email protected] (Y.M.) *E-mail: [email protected] (S.M.)

KEYWORDS: immunochromatographic test, lateral flow test, immunoassay, influenza virus, gold nanoparticles, platinum nanoparticles, localized surface plasmon resonance

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ABSTRACT

The development of a sensitive and rapid diagnostic test for early detection of infectious viruses is urgently required to defend against pandemic and infectious diseases including seasonal influenza. In this study, we developed noble metal (Au, Pt) nanoparticle–latex nanocomposite particles for use as probes for immunochromatographic test (ICT) strips. The nanocomposite particles were conjugated with monoclonal antibody (mAb) to detect an influenza A (H1N1) antigen. For comparison, Au nanoparticles conjugated with mAb were also prepared. The lowest detectable concentrations of the influenza A antigen were found to be 6.25×10−3 and 2.5×10−2 HAU/mL for Au nanoparticle–latex and Pt nanoparticle–latex nanocomposite particles, respectively, whereas it was 4.0×10−1 HAU/mL for Au nanoparticles. These results clearly demonstrated that the nanocomposite probes were more sensitive than conventional nanoparticlebased probes for ICT. To expand the versatility of the nanocomposite probes, the surfaces of the probes were functionalized with biotinylated proteins to enable modification of their surfaces with desired biotinylated antibodies through biotin–avidin binding.


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1. INTRODUCTION Highly sensitive and rapid detection of biomarkers and/or antigens is extremely important for clinical diagnostics,1,2 basic medical research,3,4 and a variety of other biomedical applications.5–10 At present, immunochromatographic tests (ICTs) are used for rapid detection of biomarkers and/or antigens in bodily fluids including saliva,11–13 urine,14–17 and blood.18–20 Compared with clinical inspection and instrumental analysis, ICTs are cost-effective, easy to use, and do not need skilled technicians.21–23 These advantages make ICTs an extremely important format for medical diagnosis, hormone testing, drug testing, and point-of-care testing, particularly for on-site patient care.24–28 Most ICT strips function based on the colorimetric method using colored particles such as colloidal gold (Au) nanoparticles,29 colored latex beads,30 quantum dots,31-33 colored silica nanoparticles34 and luminol-reduced Au nanoparticles,35,36 etc. Among the aforementioned colored particles, colloidal Au nanoparticles are widely used in ICTs because of their favorable localized surface plasmon resonance (LSPR) properties and excellent chemical stability, which enable qualitative or semiquantitative detection.37-39 However, a major limitation of current ICT systems is that their range of sensitivity is restricted to biomarkers or antigens with relatively high abundance in the samples.40 Therefore, the detection sensitivity of ICT strips needs to be improved to expand their range of applications. The probes for ICT strips strongly influence the detection sensitivity. The detection limit and sensitivity of an Au nanoparticle-based ICT strip are primarily determined by the optical extinction properties of the Au nanoparticles and their binding capacity for the target analyte.41,42 Therefore, a straightforward approach to improve the detection limit and sensitivity of Au nanoparticle-based ICT strips is to amplify the signal from Au nanoparticles via the effective aggregation of Au nanoparticles at the detection line. In reality, however, it is extremely difficult



to substantially enhance the binding capacity of Au nanoparticles for the target analyte when the analyte concentration is low. With this in mind, we conceived the idea of fabricating monodisperse Au nanoparticle–latex nanocomposites as probes. This approach readily enables one to amplify the signal from Au nanoparticles at the detection line because a single nanocomposite particle intrinsically contains multiple Au nanoparticles. Platinum (Pt) nanoparticles absorb light in the visible region because of the interband transition from the 5d band to the sp conduction band,43 and have excellent chemical stability rivaling that of Au nanoparticles. Although they are not as vivid as Au nanoparticles, Pt nanoparticles are a dark brown color that gives relatively high visual contrast. Therefore, using monodisperse Pt nanoparticle–latex nanocomposites as probes for ICT strips should improve the detection limit and sensitivity of the strips. In this study, we design latex particles consisting of poly(2-vinylpyridine) cross-linked with divinylbenzene (denoted as P2VPs) decorated with Au or Pt nanoparticles as probes for ICT strips. Those organic–inorganic hybrid nanocomposite particles are intensely colored even at low analyte concentration. The particles are labeled with antibodies using conventional techniques. In addition, the performance of the metal (Au, Pt) nanoparticle–latex nanocomposites as probes for ICT strips is examined using an influenza A antigen as a model analyte. Finally, the nanocomposites are biofunctionalized with the biotin–avidin system44,45 to enhance their versatility in biomedicine. This strategy expands the suitability of the metal nanoparticle–latex nanocomposites for various biochemical applications and will aid development of novel bioassay platforms.

2. MATERIALS AND METHODS


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2.1. Materials. 2-Vinylpyridine (2-VP) and divinylbenzene (DVB) were purchased from Wako Pure Chemical (Osaka, Japan) and treated with basic alumina before use to remove p-tertbutylcatechol. Poly(ethylene glycol) methyl ether methacrylate (PEGMA) macromonomer (Mn ~2080, 50 wt% in H2O), Aliquat 336, and biotin-4-fluorescein (b-4F) were purchased from Sigma-Aldrich (HAuCl4·4H2O),

and

used

hydrogen

as

received.

Hydrogen

hexachloroplatinate(IV)

tetrachloroaurate(III)

hexahydrate

tetrahydrate

(H2PtCl6·6H2O),

α,α’-

azodiisobutyramidine (AIBA), dimethylamine borane (DMAB), and neutralized avidin from egg white (Navi) were purchased from Wako Pure Chemical and used without further purification. Biotinylated bovine serum albumin (bBSA) was purchased from Thermo Fisher Scientific. Inactivated influenza A antigen [A/California/7/2009(H1N1)] and detection antibodies (monoclonal antibody: mAb, influenza A NP clone A56-1, and conjugated clone A43-6) were provided by ADTEC Co., Ltd (Oita, Japan). The hemagglutination assay (HA) value of inactivated influenza A antigen is 640.46 ICT strips consisting of a nitrocellulose reaction membrane, sample pad, and absorbent pad were purchased from ADTEC Co., Ltd. 2.2. Synthesis of Latex Particles (P2VPs). The P2VP latex particles used in this study were prepared according to the literature.47 Briefly, Aliquat 336 (3 g), PEGMA (10 g of a 50 wt% aqueous solution), and deionized water (300 mL) were placed in a round-bottom flask. Then, 2-VP (48 g) and DVB (2 g) were added to the solution. The reaction mixture was stirred using a mechanical stirrer and heated to 60 °C. An aqueous AIBA solution (1.4 wt%, 18 mL) as an initiator was added dropwise to the mixture after 1 h of stirring at 60 °C. The polymerization was carried out for 24 h at 60 °C under a dry nitrogen flow. The resulting milky-white colloidal dispersions were then purified by five centrifugation/redispersion cycles, replacing the



supernatant with fresh deionized water each time. The mean diameter of the resulting P2VPs measured from scanning electron microscopy (SEM) images was 356 ± 6.2 nm. 2.3. Preparation of P2VPs Decorated with Au Nanoparticles (Au-P2VPs). AuP2VPs were synthesized according to the literature with some modifications.48 The aqueous dispersion of P2VPs (3.0 wt%, 1.5 g) was mixed with HAuCl4 aqueous solution (40 mM, 8.0 mL) at room temperature. After stirring for 3 h, the free metal ions were removed by several cycles of centrifugation and redispersion in deionized water, providing precursor particles. DMAB aqueous solution (50 mM, 3.0 mL) was gradually mixed with the aqueous dispersion of precursor particles (0.067 wt%, 48.5 g) under stirring for 3 h to form Au nanoparticles on the surfaces of P2VPs, followed by dialysis using deionized water after inspissation. 2.4. Preparation of P2VPs Decorated with Pt Nanoparticles (Pt-P2VPs). The aqueous dispersion of P2VPs (3.0 wt%, 1.5 g) was mixed with H2PtCl6 aqueous solution (40 mM, 5.0 mL) at room temperature. After stirring for 3 h, the free metal ions were removed by several cycles of centrifugation and redispersion in deionized water, providing precursor particles. DMAB aqueous solution (13 mM, 14 mL) was gradually mixed with the aqueous dispersion of precursor particles (0.073 wt%, 48.2 g) under stirring for 3 h to form Pt nanoparticles on the surfaces of P2VPs, followed by dialysis using deionized water after inspissation. 2.5. Antibody Conjugation. Labeling of Au-P2VPs or Pt-P2VPs with mAb was carried out as follows: Au-P2VP or Pt-P2VP aqueous dispersion (1.0 wt%, 100 µL) and boric acid buffer (100 mM, pH=8.5, 900 µL) containing mAb (100 µg) were placed in a microtube and then mixed for 3 h at room temperature. The mixture was centrifuged at 664 ×g for 5 min and then the supernatant was discarded. Block Ace (DS Pharma Biomedical Co., Ltd., Osaka, Japan) (1.0


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wt%, 1.0 mL) was added to the precipitate, followed by sonication and stirring for 2 h at room temperature. The resulting mAb-conjugated Au-P2VPs or Pt-P2VPs were washed three times with washing buffer [50 mM Tris-HCl (pH=8.0), 0.05 wt% Triton X-100]46 and then redispersed in storage buffer [50 mM Tris-HCl (pH=7.2), 0.05 wt% Triton X-100, 0.1 wt% BSA, 5 wt% saccharose, and 0.095 wt% sodium azide]46 to give a final concentration 0.1 wt%. The mAbconjugated Au-P2VP and Pt-P2VP particles are hereafter referred to as mAb-Au-P2VPs and mAb-Pt-P2VPs, respectively. In a similar way, bBSA-conjugated Pt-P2VPs were prepared replacing mAb with bBSA for detection of a control line. The bBSA-conjugated Pt-P2VPs are hereafter referred to as control-Pt-P2VPs. 2.6. Preparation of ICT Strips. A 5.5-mm-wide ICT strip consisting of an absorbent pad (length 27.7 mm), a nitrocellulose membrane (length 25.0 mm) and a sample pad (length 25.5 mm) was prepared. The membrane was printed with a mAb test line and an avidin control line. A band of mAb was drawn on the membrane as the test line (T line) at 8.5 mm from the sample pad, while a band of avidin was drawn on the membrane as the control line (C line) 16.7 mm from the sample pad. 2.7. Detection of Influenza A Antigen. First, mAb-Au-P2VPs or mAb-Pt-P2VPs dispersed in storage buffer (0.1 wt%, 3.0 µL) and control-Pt-P2VPs dispersed in storage buffer (0.1 wt%, 3.0 µL) were mixed together with an inactivated influenza A antigen solution [100 µL, 50 mM Tris-HCl (pH=7.1), 150 mM NaCl, 1wt% BSA, 1wt% Triton X-100].46 Immediately after mixing, 50 µL of the mixture was transferred onto the ICT strip and kept still for 15 min. The optical signal value on the test line of the ICT strip was read by an immunochromato reader (Hamamatsu Photonics C10066). Au nanoparticles purchased from Tanaka Kikinzoku Kogyo



(mean diameter: 30 nm) were also treated with mAb and evaluated by the same procedure for comparison. The mAb-conjugated Au nanoparticles are hereafter referred to as mAb-AuNPs. 2.8. Biotinylated Protein Conjugation. An aqueous dispersion of Au-P2VPs or PtP2VPs (1.0 wt%, 10 µL) and bBSA aqueous solution (10 mg/mL, 10 µL) were added to deionized water (1.0 mL) and then mixed together well at room temperature for 30 min. The mixture was centrifuged at 2656 ×g for 5 min and the supernatant was discarded. BSA aqueous solution (10 mg/mL, 30 µL) and deionized water (1.0 mL) were added to the precipitate, followed by sonication and stirring for 30 min at room temperature. The mixture was washed by two centrifugation/redispersion cycles with phosphate-buffered saline (PBS; 150 mM, pH=7.1). The bBSA-conjugated Au-P2VP and Pt-P2VP particles are hereafter referred to as bBSA-AuP2VPs and bBSA-Pt-P2VPs, respectively. After purification, Navi (10 µL, 10 mg/mL in deionized water) was added to the PBS dispersion of bBSA-Au-P2VPs or bBSA-Pt-P2VPs (0.01 wt%) and mixed well at room temperature, followed by two centrifugation/redispersion cycles with PBS. The Navi-conjugated bBSA-Au-P2VP and bBSA-Pt-P2VP particles are hereafter referred to as Navi-bBSA-Au-P2VPs and Navi-bBSA-Pt-P2VPs, respectively. Subsequently, b4F ethanolic solution (1 mM, 10 µL) was added to the Navi-bBSA-Au-P2VP or Navi-bBSA-PtP2VP dispersion, followed by four centrifugation/redispersion cycles with PBS, giving a final concentration of 0.01 wt%. 2.9. Characterization. The morphology and structure of particles were characterized using an ultra-high-resolution scanning electron microscope (Hitachi SU9000) equipped with a high-angle annular dark-field (HAADF) detector and energy-dispersive X-ray spectrometer (EDS). SEM images were recorded on the SEM operating at 30 kV. Cross-sectional transmission electron microscopy (TEM) images were recorded in the scanning TEM (STEM) mode on the


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SEM operating at 30 kV. For cross-sectional TEM observation, the particles were embedded in epoxy resin, cured and then sectioned into a slice with a thickness of about 60 nm using an ultramicrotome (Leica Ultracut UCT). X-ray diffraction (XRD) patterns were recorded using an X-ray diffractometer (Rigaku MiniFlex600) operated in reflection geometry at room temperature with Cu Kα radiation. The mean sizes and size distributions of particles were estimated from SEM images and measured using an analytical centrifuge (LUM GmbH LUMiSizer 610). The measurement conditions of the analytical centrifuge were a temperature of 25 °C, path length of 2 mm, concentration of 0.02 wt%, and rotation speed of 2000 rpm. The optical properties of samples were characterized by an ultraviolet–visible (UV-vis) spectrometer (Shimadzu UV-3600 Plus) at room temperature. The zeta potential and hydrodynamic diameter of particles was measured using a Malvern Zetasizer Nano ZS ZEN3600. Confocal fluorescent images were obtained using a confocal laser scanning microscope (Olympus FV1000) connected to an IX 81 inverted microscope. Samples were excited with the 473-nm diode laser line and detected in the wavelength range of 490–590 nm.

3. RESULTS AND DISCUSSION Figure 1 shows a schematic illustration of the formation of Au-P2VPs and Pt-P2VPs by adsorption of metal ions (AuCl4− or PtCl62−) onto P2VPs followed by reduction. The P2VPs are capable of ion loading via electrostatic interactions because they have pyridine moieties, which can act as effective ion adsorption sites. After ion adsorption, the color of the P2VP dispersion changed from milky white to yellow in the case of AuCl4− or pale orange in the case of PtCl62−. EDS analyses of the ion-loaded P2VPs confirmed that Au or Pt species were present in the P2VPs (data not shown). The metal contents of the ion-loaded P2VPs were determined to be 1.9



and 1.3 mmol/g for Au and Pt, respectively, by measuring the mass of each residue after heat treatment in air for 3 h at 500 °C.

Figure 1. Schematic illustration of the synthesis of Au-P2VP and Pt-P2VP nanocomposite particles.

When DMAB solution was added to the AuCl4−-loaded P2VP dispersion to form Au-P2VPs, the color of the dispersion quickly changed from yellow to dark purple, indicating the formation of Au nanoparticles on/in the P2VPs. Conversely, when DMAB solution was added to the PtCl62−-loaded P2VP dispersion to form Pt-P2VPs, the color of the dispersion slowly changed from pale orange to black, consistent with the formation of Pt nanoparticles on/in the P2VPs. Figure 2A shows a low-magnification SEM image of Au-P2VPs. Figure 2B and C display SEM and HAADF-STEM images of the same single Au-P2VP particle, respectively. Figure 2D shows a cross-sectional HAADF-STEM image of a single Au-P2VP particle. It is clearly seen


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that the Au nanoparticles are only present near the surface of the particle. Figure 2G and H show EDS elemental mapping images for C and Au of a single Au-P2VP particle, respectively. The Au-P2VPs are uniform and spherical in shape with a mean diameter of 368.2 ± 6.7 nm (Figure 2I) and possess a core–satellite structure in which P2VP cores are uniformly decorated with Au nanoparticles. These observations also clearly demonstrate that Au nanoparticles with a mean diameter of 23.0 ± 4.7 nm (Figure 2J) were uniformly formed near the surface of the spherical P2VP particles.

Figure 2. SEM images of (A) ensemble of Au-P2VPs and (B) a single Au-P2VP. HAADFSTEM images of (C) the single Au-P2VP in (B) and (D) a cross-sectional slice of another single Au-P2VP. (E, F) Magnified images of B and C, respectively. (G, H) EDS elemental mapping images of the single Au-P2VP in (B). (G) C K-line and (H) Au L-line. Size histograms of (I) AuP2VPs and (J) Au nanoparticles on P2VPs.



Taking a closer look at each of the Au nanoparticles on a single Au-P2VP particle, it is found that the individual Au nanoparticles appear slightly larger in the HAADF-STEM image (Figure 2F) than in the SEM image (Figure 2E). In particular, some Au nanoparticles overlap with each other in the HAADF-STEM image despite being able to be resolved independently in SEM images, as indicated by arrows in Figure 2E and F. This discrepancy is because only the exposed portion of the Au nanoparticles on the P2VP surface is observed in the SEM image, whereas the entire Au nanoparticles, including the portion buried in the P2VP particle, are observed in the HAADF-STEM image. Therefore, these observations reveal that part of each Au nanoparticle is buried in the P2VP particle (Supporting Information Figure S1). Figure 3A–C show a low-magnification SEM image of an ensemble of Pt-P2VPs and SEM and HAADF-STEM images of a single Pt-P2VP particle, respectively. Figure 3D depicts the cross-sectional HAADF-STEM image of a single Pt-P2VP particle. As is the case for the AuP2VPs, the Pt nanoparticles are mainly formed near the surface of the P2VP particles. However, a non-negligible number of Pt nanoparticles were found to exist in high density in the subsurface region (around 70 nm from the surface of the P2VP particle). Figure 3G and H show EDS elemental mapping images for C and Pt of a single Pt-P2VP particle. The Pt-P2VPs are uniform and spherical in shape with a rough surface and mean diameter of 368.2 ± 7.6 nm (Figure 3I). Each Pt-P2VP particle has numerous small Pt nanoparticles with a mean diameter of 3.8 ± 1.4 nm (Figure 3J), which was much smaller than the Au nanoparticles of Au-P2VP. Moreover, a greater number of Pt nanoparticles can be seen in the HAADF-STEM image (Figure 3F) than in the SEM image (Figure 3E). These observations also suggest that Pt nanoparticles are formed not only on the surface of P2VP particles but also inside the P2VP core.


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The Au or Pt nanoparticles obtained after chemical reduction adhered strongly to the P2VPs; the Au or Pt nanoparticles did not detach from the P2VPs even after shaking or sonication. We believe that Au or Pt nanoparticles formed on the P2VP surface may adhere to the P2VP core latex particles through the anchor effect described above in addition to the chemical interactions of the metal nanoparticles with the pyridine moieties of the P2VPs.

Figure 3. SEM images of (A) an ensemble of Pt-P2VPs and (B) a single Pt-P2VP. HAADFSTEM images of (C) the single Pt-P2VP in (B) and (D) a cross-sectional slice of another single Pt-P2VP. (E, F) Magnified images of B and C, respectively. (G, H) EDS elemental mapping images of the single Pt-P2VP in (B). (G) C K-line and (H) Pt L-line. Size histograms of (I) PtP2VPs and (J) Pt nanoparticles on P2VPs.

The metal contents of Au-P2VPs and Pt-P2VPs determined by measuring the mass of the residue after heat treatment in air for 3 h at 500 °C were 48.1 and 35.5 wt%, respectively. Based



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on these results, the number of metal nanoparticles loaded in a single P2VP, n, was calculated to be 208 for Au-P2VPs and 25,143 for Pt-P2VPs according to the following equation:

݊=

ܸߩ߮ ‫ߩݒ‬୫ୣ୲ୟ୪

(1)

1 ߮ 1−߮ = + ߩ ߩ୫ୣ୲ୟ୪ ߩ୮୭୪୷୫ୣ୰

(2)

where V is a volume of a single Au-P2VP (or Pt-P2VP) particle, v is a volume of a single Au (or Pt) nanoparticle, ρ is the average density of Au-P2VPs (or Pt-P2VPs), ρmetal is the density of Au (or Pt), ρpolymer is the density of P2VP, and φ is the mass fraction of Au (or Pt). The size distributions of particles obtained via the analytical centrifugation technique are shown in Figure 4. The mean sizes of Au-P2VPs and Pt-P2VPs are determined to be 362.4 ± 11.4 and 381.8 ± 12.6 nm, respectively. These results are consistent with the particle sizes measured from SEM images. Moreover, this result confirms that both Au-P2VPs and Pt-P2VPs are monodisperse and colloidally stable. Au-P2VPs and Pt-P2VPs displayed high dispersion stability even at a concentration of 1 wt%. To further assess their colloidal stability, we analyzed the size distributions of Au-P2VPs and Pt-P2VPs after refrigerated storage in aqueous solutions for 8 months. The size distributions of Au-P2VPs and Pt-P2VPs did not change after this period, confirming that they also have excellent long-term dispersion stability (data not shown). XRD patterns of P2VPs, Au-P2VPs, and Pt-P2VPs are shown in Figure 5. In the case of AuP2VPs, the diffraction peaks observed at 2θ = 38.17°, 44.25°, 64.60°, 77.61°, and 81.78° corresponded to the (111), (200), (220), (311), and (222) planes of face-centered cubic (fcc) Au, respectively. In the case of Pt-P2VPs, the peaks detected at 2θ = 39.86°, 46.19°, 67.52°, 81.50°, and 85.41° corresponded to the (111), (200), (220), (311), and (222) planes of fcc Pt, respectively. The mean crystallite sizes of Au and Pt estimated from the (111) peak using the


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Scherrer formula were 9.3 and 2.7 nm, respectively. These results suggest that Pt nanoparticles on/in Pt-P2VPs are single crystalline, whereas the Au nanoparticles on/in Au-P2VPs are polycrystalline in nature.

Figure 4. Particle size distributions of (A) Au-P2VPs and (B) Pt-P2VPs dispersed in deionized water (0.02 wt%) at 25 °C measured using an analytical centrifuge.

Figure 5. XRD patterns of (A) Au-P2VPs, (B) Pt-P2VPs, and (C) P2VPs. The reference patterns shown in the figure are fcc Au (blue line, JCPDS PDF no. 01-071-4615) and fcc Pt (red line, PDF no. 00-004-0802).



Figure 6 shows the pH dependence of the zeta potential of P2VPs, Au-P2VPs, and Pt-P2VPs dispersed in water. The zeta potential of Au-P2VPs was positive in the pH range of 3–10 and its maximum value was about +40 mV around pH=5 (Figure 6B). No isoelectric point (IEP) was observed. The pH dependence of the zeta potential of Au-P2VPs was quite similar to that of P2VPs (Figure 6A). In contrast, Pt-P2VPs displayed an IEP at pH=5.7 and showed negative zeta potential values (ca. −50 mV) at basic pH. This result suggests that the molecular structure of P2VPs in Pt-P2VPs might be altered by the formation of Pt nanoparticles.

Figure 6. Zeta potentials of (A) P2VPs, (B) Au-P2VPs, and (C) Pt-P2VPs.

To gain more insight into the amphoteric nature of Pt-P2VPs, we measured the zeta potentials of Au/Pt-P2VPs fabricated with different Au/Pt input molar ratios (Figure S2). With decreasing Au/Pt input molar ratio, the zeta potential systematically changed from that of AuP2VPs to that of Pt-P2VPs. Moreover, solid-state

13

C NMR spectra of P2VPs, Au-P2VPs, and

Pt-P2VPs were recorded, as shown in Figure S3. Peaks originating from aromatic rings and poly(ethylene glycol) (PEG) appeared at 110–170 and 69.5 ppm, respectively. The integral ratio of the PEG part to the aromatic part (PEG/aromatic) was calculated for P2VPs, Au-P2VPs, and Pt-P2VPs. The value of PEG/aromatic for Au-P2VPs was identical to that of P2VPs, as shown in Table S1, whereas that of Pt-P2VPs was 27% lower than those of P2VPs and Au-P2VPs. These


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results strongly suggest that carboxyl groups are formed on the surface of Pt-P2VPs through hydrolysis of ester groups, which is probably driven by the catalytic effect of Pt complexes.49 Figure 7 shows UV-vis spectra and photographs of Au-P2VP and Pt-P2VP aqueous dispersions. The dispersions of P2VPs, Au-P2VPs, and Pt-P2VPs were cloudy white, deep purple, and dark brown, respectively. The UV-vis spectrum of the Au-P2VP dispersion showed an LSPR band at 591 nm, which is markedly redshifted from the LSPR peak of pure Au nanoparticles (AuNPs) with a mean diameter of 30 nm50 at 520 nm. This redshift is presumably caused by the plasmon coupling between Au nanoparticles densely embedded near the surface of the P2VP particles.

Figure 7. UV-vis spectra of Au-P2VPs (blue curve) and Pt-P2VPs (red curve). For comparison, the UV-vis spectra of P2VPs (gray curve) and AuNPs (green curve) are also plotted. The inset shows photographs of aqueous dispersions of Au-P2VPs and Pt-P2VPs. Note that the numbers of particles are the same for the cases of Au-P2VPs, Pt-P2VPs and P2VPs (1×109). In the case of AuNPs, the number of particles is 1×1011.

The LSPR extinction of Au nanoparticles usually strongly depends on the size, shape, interparticle distance, and relative permittivity of the surrounding medium. The reason for the



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redshifted broad LSPR band of Au-P2VPs would be the close-contact dipole coupling of LSPR between Au nanoparticles on an Au-P2VP particle. As seen in Figure 2E, Au nanoparticles on an Au-P2VP particle were located adjacent to each other. In fact, the average interparticle distance between Au nanoparticles on the P2VP surface was estimated to be 20.4 nm from the surface area of a P2VP particle and mean diameter of Au nanoparticles, which was smaller than the average diameter of the Au nanoparticles (23.0 ± 4.7 nm). The light extinction of a single AuP2VP (or Pt-P2VP) particle in the visible range, E, was calculated as

‫=ܧ‬

‫ߩܸܣ‬ ߮

(3)

where A is the integrated absorbance from 380–750 nm. The value of E of a single Au-P2VP particle was approximately 200 times higher than that of a single Au nanoparticle with a mean diameter of 30 nm. The UV-vis spectrum of the Pt-P2VP dispersion was featureless, as shown in Figure 7, because Pt nanoparticles do not have an LSPR band within the visible range. However, Pt-P2VPs displayed light absorption capability over the entire visible range, so the light extinction of Pt-P2VPs was higher than that of P2VPs. The value of E of a single Pt-P2VP was calculated to be approximately 100 times higher than that of a single Au nanoparticle with a mean diameter of 30 nm. Therefore, both Au-P2VPs and Pt-P2VPs were used in the following ICT experiments. As illustrated in Figure 8, Au-P2VPs and Pt-P2VPs were used to detect influenza A antigen via ICT. Because Au-P2VPs and Pt-P2VPs have Au and Pt nanoparticles on their surfaces, respectively, antibodies can be easily conjugated on the surfaces by physical coupling in the same manner as for colloidal gold. To confirm the successful conjugation of mAb on the surface of Au-P2VPs and Pt-P2VPs, the zeta potential and hydrodynamic diameter of Au-P2VPs and PtP2VPs were measured before and after treatment with mAb, and, subsequent after treatment with 18 Environment ACS Paragon Plus

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Block Ace (Figures S4 and S5). The zeta potential of Au-P2VPs in the pH range of 7.5–10 changed from positive to negative and its minimum value changed from +10 mV (Figure 6B) to –33 mV (Figure S4A) following conjugation with mAb. Au-P2VPs displayed an IEP at pH=7.4 after treatment with mAb as shown in Figure S4A. After subsequent treatment with Block Ace, the IEP shifted to pH=5.2 (Figure S4B). On the other hand, the average hydrodynamic size of Au-P2VPs increased from 420 to 690 nm after treatment with mAb and then it returned to 440 nm after treatment with Block Ace as shown in Figure S5A. The hydrodynamic size was varied in response to the change of the IEP. In the case of Pt-P2VPs, the IEP slightly shifted from 5.7 (Figure 6C) to 5.5 (Figure S4C). After subsequent treatment with Block Ace, the IEP shifted to pH=4.9 (Figure S4D). Therefore, the hydrodynamic size of Pt-P2VPs (~400−500 nm) did not vary significantly as shown in Figure S5B. Those results indicate the conjugation of antibody with Au-P2VPs and Pt-P2VPs was successful.

Figure 8. Schematic illustration of the assembly of the ICT strip. mAb-Au-P2VPs (or mAb-PtP2VPs) and control-Pt-P2VPs were mixed together with an inactivated influenza A antigen solution. Immediately after mixing, 50 µL of the mixture was transferred onto the ICT strip.



The ICT detection results for influenza A antigen with different concentrations [dilution of the original influenza A antigen solution (640 HAU/mL) by 1.0×102 to 1.024×105 times] are presented in Figure 9. In this study, the evaluation was conducted either by the naked eye or with a strip reader that read intensity. In the absence of antigen, no color change was observed because no mAb-Au-P2VP/antigen or mAb-Pt-P2VP/antigen complexes were captured on the test line. The colors of the test lines of mAb-Au-P2VPs (dark blue, top row in Figure 9A) and mAb-Pt-P2VPs (dark brown, middle row in Figure 9A) were visible down to dilutions of 1.024×105 and 2.56×104 times, respectively. In contrast, the color of the test line of mAb-AuNPs was only visible down to a dilution of 1.6×103 times (wine red, bottom row in Figure 9A).

Figure 9. (A) A series of dilutions of an influenza A antigen were tested with immunochromatographic strips [dilution of the original influenza A antigen solution (640 HAU/mL) of 1.0×102 to 1.024×105 times] using (top) mAb-Au-P2VPs, (middle) mAb-Pt-P2VPs, and (bottom) mAb-AuNPs. The left column shows color photographs of the strips and the right column shows black and white negative images of the photographs with enhanced contrast (shown for clarity). NC, C line and T line denote negative control, control line and test line, respectively. (B) Absorbance values at the test line plotted as a function of antigen dilution.


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The absorbance values of the test line were recorded, as shown in Figure 9B. The absorbance values decreased with antigen concentration, consistent with the visual evaluation. The ICT strips using mAb-Au-P2VPs or mAb-Pt-P2VPs exhibited higher sensitivity than that of mAb-AuNPs for qualitatively detecting an influenza A antigen, particularly at low antigen concentrations. Figure 10 shows absorbance values as a function of evaluation time using different labels (×400 dilution). In the case of mAb-AuNPs, equilibrium was not achieved after evaluation for 15 min and the absorbance value was only 54×10−3. In the cases of mAb-Au-P2VPs and mAb-PtP2VPs, absorbance values increased quickly during evaluation and reached saturation at ca. 570×10−3 and ca. 370×10−3, respectively. Moreover, even after 5 min, mAb-Au-P2VPs and mAb-Pt-P2VPs showed absorbance values that were respectively 9 and 3 times higher than that of mAb-AuNPs. Therefore, the ICT strips with mAb-Au-P2VPs or mAb-Pt-P2VPs enabled detection of an influenza A antigen much faster than that with mAb-AuNPs.

Figure 10. Plots of the absorbance on the test line as a function of evaluation time (×400 antigen dilution). Blue, red, and green represent mAb-Au-P2VPs, mAb-Pt-P2VPs, and mAb-AuNPs, respectively.



Conjugation of biotinylated proteins to the Au-P2VP and Pt-P2VP surfaces should enable their use in various biomedical applications. Therefore, the surfaces of Au-P2VPs and Pt-P2VPs were functionalized with bBSA (giving bBSA-Au-P2VPs and bBSA-Pt-P2VPs, respectively) to expand the versatility of these nanocomposite probes, because desired biotinylated antibodies can be easily conjugated to the surfaces of such probes depending on the target antigen using avidin as an intermediary. Figure 11 shows the pH dependence of the zeta potential of bBSA-AuP2VPs and bBSA-Pt-P2VPs dispersed in water. The zeta potential of Au-P2VPs in the pH range of 5.2–10 changed from positive to negative and its minimum value changed from +10 mV (Figure 6B) to −50 mV (Figure 11A) following functionalization with bBSA. In the case of PtP2VPs, the IEP shifted from 5.7 (Figure 6C) to 5.0 (Figure 11B) and the minimum value of the zeta potential changed from −50 mV (around pH=10 in Figure 6C) to −60 mV (around pH=7 in Figure 11B) after functionalization with bBSA.

Figure 11. Zeta potentials of (A) bBSA-Au-P2VPs and (B) bBSA-Pt-P2VPs.

From the above results, it is considered that the zeta potential changed upon the coating of bBSA on the surface of Au-P2VPs and Pt-P2VPs. In addition, the particle sizes were also measured by dynamic light scattering (Malvern Zetasizer Nano ZS ZEN3600). The average sizes


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of Au-P2VPs increased from 415 to 728 nm and that of Pt-P2VPs from 432 to 511 nm after bBSA functionalization. These results indicate the formation of bBSA-Au-P2VPs and bBSA-PtP2VPs. To further confirm that bBSA was successfully conjugated on the surface of Au-P2VPs and Pt-P2VPs to form bBSA-Au-P2VPs and bBSA-Pt-P2VPs, respectively, fluorescence microscopy observations were conducted. First, bBSA-Au-P2VPs and bBSA-Pt-P2VPs were conjugated with Navi to form Navi-bBSA-Au-P2VPs and Navi-bBSA-Pt-P2VPs, respectively. Then, Navi-bBSA-Au-P2VPs and Navi-bBSA-Pt-P2VPs were treated with b-4F, which selectively binds to Navi, as shown in Figure 12A. As a control experiment, bBSA-Au-P2VPs and bBSA-Pt-P2VPs were directly treated with b-4F, as presented in Figure 12B.

Figure 12. Schematic illustration of the verification of bBSA modification of Au-P2VPs and PtP2VPs using biotin–avidin binding. Processes (A) with and (B) without Navi treatment. If b-4F strongly binds to Au-P2VPs or Pt-P2VPs, one can detect fluorescence from the particles, whereas no fluorescence will be observed from the particles if b-4F does not bind to them.



As shown in Figure 13, strong fluorescence was observed from Navi-bBSA-Au-P2VPs and Nabi-bBSA-Pt-P2VPs after b-4F treatment, but not from bBSA-Au-P2VPs and bBSA-Pt-P2VPs. These results unambiguously confirmed that the surfaces of the Au-P2VPs and Pt-P2VPs were successfully functionalized with bBSA. In addition, these findings also confirmed the feasibility of on-demand biofunctionalization (such as with antibodies, ligands, or DNA) of the nanocomposite probes via the biotin–avidin strategy.

Figure 13. Transmission (left column), fluorescent (middle column), and merged (right column) images of (A) Navi-bBSA-Au-P2VPs, (B) bBSA-Au-P2VPs, (C) Navi-bBSA-Pt-P2VPs, and (D) bBSA-Pt-P2VPs after b-4F treatment. All scale bars are 5 µm.


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4. CONCLUSIONS We synthesized latex particles uniformly decorated with Au or Pt nanoparticles through emulsion polymerization and direct metallization. The light extinction of a single Au-P2VP was 200 times higher than that of a single Au nanoparticle with a mean diameter of 30 nm, whereas that of a single Pt-P2VP was 100 times higher than that of a single Au nanoparticle. This was caused by the nanocomposite structure in which a large number of Au or Pt nanoparticles were immobilized on the surface of each P2VP latex microparticle. Because of their enhanced light extinction, Au-P2VPs and Pt-P2VPs can be used as high-performance probes for ICT. To demonstrate their performance, an influenza antigen (H1N1) was detected using Au-P2VPs and Pt-P2VPs as probes for ICT strips. The naked-eye detection sensitivities were enhanced by more than 64- and 16-fold for the strips with Au-P2VPs and Pt-P2VPs, respectively, compared with that using conventional colloidal gold. Moreover, we also functionalized the surfaces of AuP2VPs and Pt-P2VPs with biotinylated proteins. Our study revealed that Au-P2VPs and PtP2VPs are attractive as versatile probes for various applications in the fields of biology and biomedicine.

ASSOCIATED CONTENT Supporting Information. Schematic diagram of an Au nanoparticle buried in P2VP (Figure S1); zeta potential graphs of the metal (Au, Pt) nanoparticle–latex nanocomposites (Figure S2); solid-state

13

C NMR spectra of the nanocomposites (Figure S3); integral ratios of functional



13

C NMR spectra (Table S1); zeta potential graphs of the

groups estimated from solid-state antibody-conjugated

metal

Page 26 of 48

(Au,

Pt)

nanoparticle–latex

nanocomposites

(Figure

S4);

hydrodynamic diameter distributions of the metal (Au, Pt) nanoparticle–latex nanocomposites before and after treatment with antibody.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (Y.M.) *E-mail: [email protected] (S.M.)

Author Contributions The manuscript was written through contributions from all authors. All authors have approved the final version of the manuscript.

Notes The authors report no conflict of interest.

ACKNOWLEDGMENTS The authors thank ADTEC Co., Ltd. for providing of influenza antigen and antibody. We thank Natasha Lundin, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.


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TOC Graphic


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Figure 1 508x352mm (125 x 125 DPI)

ACS Paragon Plus Environment


Figure 2 232x179mm (180 x 180 DPI)


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Figure 3 231x180mm (180 x 180 DPI)



Figure 4 502x162mm (147 x 147 DPI)


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Figure 5 210x250mm (150 x 150 DPI)



Figure 6 500x136mm (150 x 150 DPI)


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Figure 7 260x190mm (256 x 256 DPI)



Figure 8 201x186mm (256 x 256 DPI)


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Figure 9 527x234mm (148 x 148 DPI)



Figure 10 269x190mm (256 x 256 DPI)


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Figure 11 470x186mm (150 x 150 DPI)



Figure 12 348x263mm (150 x 150 DPI)


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Figure 13 202x248mm (160 x 160 DPI)



TOC Graphic 508x216mm (150 x 150 DPI)


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