An Antibody-Immobilized Silica Inverse Opal Nanostructure for Label-Free Optical Biosensors

Three-dimensional SiO2-based inverse opal (SiO2-IO) nanostructures were prepared for use as biosensors. SiO2-IO was fabricated by vertical deposition and calcination processes. Antibodies were immobilized on the surface of SiO2-IO using 3-aminopropyl trimethoxysilane (APTMS), a succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] ester (NHS-PEG4-maleimide) cross-linker, and protein G. The highly accessible surface and porous structure of SiO2-IO were beneficial for capturing influenza viruses on the antibody-immobilized surfaces. Moreover, as the binding leads to the redshift of the reflectance peak, the influenza virus could be detected by simply monitoring the change in the reflectance spectrum without labeling. SiO2-IO showed high sensitivity in the range of 103–105 plaque forming unit (PFU) and high specificity to the influenza A (H1N1) virus. Due to its structural and optical properties, SiO2-IO is a promising material for the detection of the influenza virus. Our study provides a generalized sensing platform for biohazards as various sensing strategies can be employed through the surface functionalization of three-dimensional nanostructures.


Introduction
Influenza is an acute infectious disease caused by the influenza virus. The virus, which belongs to a genus of the Orthomyxoviridae family, is divided into three types: influenza A, B, and C [1]. The influenza A virus has recurrent epidemics and is recognized as a serious public health hazard [2]. A rapid and precise diagnosis is therefore important to prevent the spread of the disease. Existing virus detection techniques, such as enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR), have drawbacks, such as requiring time-consuming and specialized processes [3][4][5][6]. Therefore, a technique that is convenient, rapid, sensitive and selective is required for the detection of pandemic viruses. Recently, diverse integrated optical biosensors, based on interferometers, grating couplers, microring resonators, photonic crystals, or micro/nanophotonics transducers, have been studied for the detection of various bio-markers [7,8]. These techniques can be utilized to produce highly-sensitive and ultra-compact biosensors using light-matter interactions [9,10]. Additionally, because integrated optical biosensors have the advantage of direct, real-time, and label-free detection, they are appropriate for the detection of biohazards such as viruses and bacteria. Among integrated

Materials and Reagents
To fabricate the IO nanostructures and perform surface functionalization, ethanol (EtOH) and methanol (MeOH) were purchased from EMD Millipore Co. To functionalize the gold nanoparticles (AuNPs), gold (III) chloride trihydrate (HAuCl4) and sodium citrate were purchased from Sigma (St. Louis, MO, USA), and gold binding peptide-protein G (GBP-proG) was purchased from Bioprogen Co. (Daejeon, Korea).

Preparation of the SiO 2 -IO Nanostructures
The SiO 2 -IO nanostructures were prepared by following the previous literature [31]. The silicon wafer substrate was cleaned by washing with EtOH, followed by immersion in piranha solution, a mixture of sulfuric acid (H 2 SO 4 ), and hydrogen peroxide (H 2 O 2 ) in a 3:1 ratio for 30 min and then in 5% hydrogen fluoride for 15 min. After washing the wafer, it was vertically immersed into the colloidal suspension. The suspension was composed of a 300-nm colloidal polystyrene (PS) aqueous solution and a hydrolyzed TEOS solution. The TEOS solution consisted of TEOS, 0.1 M HCl, and EtOH in a weight ratio of 1:1:1.5. The colloidal suspension was evaporated slowly at 65 • C for 3 days. Then, the stacked colloidal PS on the substrate was removed by calcination in a furnace at 500 • C for 2 h.

Characterization of the SiO 2 -IO Nanostructures
The morphology of SiO 2 -IO nanostructures was analyzed by scanning electron microscopy (SEM, Quanta 250 FEG, FEI, Hillsboro, OR, USA) with an acceleration voltage of 10 kV after applying an Au coating. The surface wettability of opal and the IO nanostructure was measured by a contact angle analyzer (Phoenix 300 Plus, SEO Co., Ltd., Suwon, Korea). The reflectance was measured by a spectrometer (FLAME-S, Ocean Optics, Largo, FL, USA). The spectrometer was fixed on an optical table, and the reflection was calibrated by a total reflection mirror, which reflects 100% of the light from 400 to 750 nm.

Surface Functionalization of SiO 2 -IO for Antibody Immobilization
Surface functionalization was performed through coating with APTMS, conjugation with a cross-linker, and immobilization of antibodies. The first SiO 2 -IO nanostructures were functionalized with APTMS by amine group exposure [32]. SiO 2 -IO nanostructures with exposed hydroxyl groups were immersed in 0.2% ATPMS solution in anhydrous toluene under a nitrogen atmosphere for 12 h. The IO nanostructures were then washed sequentially with toluene, a mixture of toluene and methanol, and methanol. After drying at 80 • C for 30 min to remove the methanol, the second IO nanostructure was conjugated by NHS-PEG 4 -maleimide (19 mM in PBS) for 30 min at room temperature to create cross-links between the protein and the nanostructures. Then, 0.1 mg/mL of Cys-ProG was conjugated to the surface by a maleimide linker for 60 min. Finally, the influenza A (H1N1) virus capture antibody (αHA-1, 1 µg/mL, 200 µL) was applied to the Cys-ProG-conjugated IO surface for 2 h. The sample was washed with PBS buffer between steps.

Detection of the Influenza Virus by the SiO 2 -IO Nanostructures
To detect the influenza A (H1N1) virus using SiO 2 -IO nanostructures, 10 µL of the virus solution was added to the αHA-1 antibody-immobilized Ab-SiO 2 -IO for 2 h. After washing with PBS buffer to remove unreacted viruses, the variation in the reflectance of the SiO 2 -IO nanostructures was measured. No blocking process was applied to the surface before virus detection. Viruses did not directly bind to the Cys-ProG-immobilized SiO 2 -IO nanostructures [21]. Additionally, we confirmed the specificity of the functionalized inverse opal structures. We treated the structure with the H1N1 subtype, the H3N2 subtype, and IFVB, and measured the reflectance change.
To confirm detection of the virus, we prepared αHA-1-functionalized AuNPs. We synthesized AuNPs by the citrate reduction method with slight modifications [33]. Then, 900 µL of the AuNPs (18 nm) were mixed with 100 µL of the GBP-ProG complex (1 mg/mL) overnight at room temperature. The solution was centrifuged twice at 12,000× rpm for 10 min for the washing process. The pellet was dispersed in 0.1× PBS buffer (containing 0.01% Tween20 buffer). In addition, 100 µL of the antibody (0.1 mg/mL) was conjugated with 900 µL of the functionalized AuNPs for 2 h at room temperature. After washing, the antibody-immobilized AuNPs were treated to confirm the virus detection of the IO nanostructures for 2 h. After rinsing with PBS, the SiO 2 -IO nanostructures were analyzed by scanning electron microscopy (SEM, Quanta 250 FEG, FEI, Hillsboro, OR, USA).

Morphologies of the SiO 2 -IO Nanostructures
IO nanostructures were generally prepared using an infiltration method in which the matrix materials, such as liquid- [25,34] or gas-phase [35][36][37] precursors, are infiltrated into the interstitial voids of colloidal arrays. After solidification of the matrix, the colloidal particles were removed by heat treatment or chemical etching. However, this method has disadvantages such as the formation of cracks, vacancies, and other defects. To overcome these drawbacks, we used the co-assembly deposition method in which colloidal PS beads and matrix precursors were simultaneously employed to form a film. Figure 1a shows the scheme for the preparation of the SiO 2 -IO nanostructures. First, a colloidal dispersion of PS beads containing TEOS was vertically deposited on the silicon (Si) substrate. When evaporated at 65 • C, the PS beads were periodically arranged to form a closely-packed array by capillary force on the Si substrate, whereas TEOS filled the interstices among the PS beads by forming solid SiO 2 through a sol-gel process. In general, cracking in opal or IO nanostructures is caused by local capillary forces during drying [38]. However, the TEOS-based sol-gel process can reduce the number of defects by forming a network between the spheres [31,39]. Then, the colloidal PS beads were removed by calcination at 500 • C. The PS beads were replaced by air in the structure, forming the IO nanostructures. To confirm detection of the virus, we prepared αHA-1-functionalized AuNPs. We synthesized AuNPs by the citrate reduction method with slight modifications [33]. Then, 900 µ L of the AuNPs (18 nm) were mixed with 100 µ L of the GBP-ProG complex (1 mg/mL) overnight at room temperature. The solution was centrifuged twice at 12,000× rpm for 10 min for the washing process. The pellet was dispersed in 0.1× PBS buffer (containing 0.01% Tween20 buffer). In addition, 100 µ L of the antibody (0.1 mg/mL) was conjugated with 900 µ L of the functionalized AuNPs for 2 h at room temperature. After washing, the antibody-immobilized AuNPs were treated to confirm the virus detection of the IO nanostructures for 2 h. After rinsing with PBS, the SiO2-IO nanostructures were analyzed by scanning electron microscopy (SEM, Quanta 250 FEG, FEI, Hillsboro, OR, USA).

Morphologies of the SiO2-IO Nanostructures
IO nanostructures were generally prepared using an infiltration method in which the matrix materials, such as liquid- [25,34] or gas-phase [35][36][37] precursors, are infiltrated into the interstitial voids of colloidal arrays. After solidification of the matrix, the colloidal particles were removed by heat treatment or chemical etching. However, this method has disadvantages such as the formation of cracks, vacancies, and other defects. To overcome these drawbacks, we used the co-assembly deposition method in which colloidal PS beads and matrix precursors were simultaneously employed to form a film. Figure 1a shows the scheme for the preparation of the SiO2-IO nanostructures. First, a colloidal dispersion of PS beads containing TEOS was vertically deposited on the silicon (Si) substrate. When evaporated at 65 °C, the PS beads were periodically arranged to form a closely-packed array by capillary force on the Si substrate, whereas TEOS filled the interstices among the PS beads by forming solid SiO2 through a sol-gel process. In general, cracking in opal or IO nanostructures is caused by local capillary forces during drying [38]. However, the TEOS-based sol-gel process can reduce the number of defects by forming a network between the spheres [31,39]. Then, the colloidal PS beads were removed by calcination at 500 °C. The PS beads were replaced by air in the structure, forming the IO nanostructures. The morphology of the fabricated opal and SiO2-IO nanostructures was characterized by SEM. Figure 1b shows the honeycomb arrangement of colloidal PS beads, which corresponds to (111) planes of a face-centered-cubic (FCC) structure. The silica precursor was solidified by a sol-gel process during the vertical deposition accompanied by evaporation at a constant temperature. During the subsequent calcination step, the PS beads were completely removed, while the SiO2 matrix The morphology of the fabricated opal and SiO 2 -IO nanostructures was characterized by SEM. Figure 1b shows the honeycomb arrangement of colloidal PS beads, which corresponds to (111) planes of a face-centered-cubic (FCC) structure. The silica precursor was solidified by a sol-gel process during the vertical deposition accompanied by evaporation at a constant temperature. During the subsequent calcination step, the PS beads were completely removed, while the SiO 2 matrix Sensors 2018, 18, 307 5 of 10 remained undistorted, as seen in Figure 1c. The cavity diameter of the SiO 2 -IO nanostructures was approximately 263.6 ± 12.9 nm, which is smaller than the size of colloidal PS beads, indicating that structural shrinkage of the SiO 2 matrix occurred during the calcination. The thickness of the SiO2-IO nanostructures on the substrate was 2.46 µm with approximately 12 layers of spherical cavities (Figure 1d). These SEM images indicated that SiO 2 based IO nanostructures were successfully fabricated by the co-assembly method.

Optical and Surface Properties of the SiO 2 -IO Nanostructures
The optical properties of IOs vary depending on the refractive index and filling factor of the medium. The stop band wavelength, or the reflectance peak position, of the IO nanostructure could be estimated by Bragg's law for (111) stacked planes of the FCC lattice [40].
where d is the (111) plane spacing, D is the particle diameter, and n e f f is the effective refractive index. ; the cavity diameter was decreased by calcination as we had discussed [41]. In the reflectance spectra experimentally measured, the opal nanostructures showed a reflectance peak at 710 nm and the IO nanostructure showed a peak at 447 nm, as shown in Figure 2a, which are comparable with the stop band positions anticipated from the Bragg's law. It is also clearly shown that the opal structure is faint gray as the peak is located in near-infrared and the IO structure is shown in sky blue (see the optical images in Figure 2a). Because the reflectance peak position can be varied depending on the refractive index of the surrounding environment, the SiO 2 -IO nanostructures can serve as sensing materials. To confirm the influence of refractive index on the reflectance spectrum, SiO 2 -IO nanostructures were infiltrated with a set of glycerin-water mixtures. Glycerin is a simple polyol compound and viscous liquid, and the refractive index of the mixture depends on the glycerin-to-water ratio. The refractive index increases as the weight percent of glycerin increases [42]; the refractive indices were 1.33, 1.36, 1.38, and 1.41 for 0, 20, 40, and 60 weight percent of glycerin, respectively. Figure S1 shows a correlation between the refractive index of the infiltration liquids and the reflectance peak position. The reflectance peak was redshifted along with the refractive index of the mixture for of the IO with constant volume and cavity size.
Sensors 2018, 18, 307 5 of 10 remained undistorted, as seen in Figure 1c. The cavity diameter of the SiO2-IO nanostructures was approximately 263.6 ± 12.9 nm, which is smaller than the size of colloidal PS beads, indicating that structural shrinkage of the SiO2 matrix occurred during the calcination. The thickness of the SiO2-IO nanostructures on the substrate was 2.46 µ m with approximately 12 layers of spherical cavities (Figure 1d). These SEM images indicated that SiO2 based IO nanostructures were successfully fabricated by the co-assembly method.

Optical and Surface Properties of the SiO2-IO Nanostructures
The optical properties of IOs vary depending on the refractive index and filling factor of the medium. The stop band wavelength, or the reflectance peak position, of the IO nanostructure could be estimated by Bragg's law for (111) stacked planes of the FCC lattice [40].
where d is the (111) plane spacing, D is the particle diameter, and is the effective refractive index.
is ; the cavity diameter was decreased by calcination as we had discussed [41]. In the reflectance spectra experimentally measured, the opal nanostructures showed a reflectance peak at 710 nm and the IO nanostructure showed a peak at 447 nm, as shown in Figure 2a, which are comparable with the stop band positions anticipated from the Bragg's law. It is also clearly shown that the opal structure is faint gray as the peak is located in nearinfrared and the IO structure is shown in sky blue (see the optical images in Figure 2a). Because the reflectance peak position can be varied depending on the refractive index of the surrounding environment, the SiO2-IO nanostructures can serve as sensing materials. To confirm the influence of refractive index on the reflectance spectrum, SiO2-IO nanostructures were infiltrated with a set of glycerin-water mixtures. Glycerin is a simple polyol compound and viscous liquid, and the refractive index of the mixture depends on the glycerin-to-water ratio. The refractive index increases as the weight percent of glycerin increases [42]; the refractive indices were 1.33, 1.36, 1.38, and 1.41 for 0, 20, 40, and 60 weight percent of glycerin, respectively. Figure S1 shows a correlation between the refractive index of the infiltration liquids and the reflectance peak position. The reflectance peak was redshifted along with the refractive index of the mixture for of the IO with constant volume and cavity size.  We also measured the contact angle to determine the wettability of the solid surface of the opal and SiO 2 -IO nanostructures. The contact angle is influenced by the surface property of the nanostructures. The contact angle on the opal structure was 82.9 • due to hydrophobic colloidal PS beads on the surface of the structure, while that on the SiO 2 -IO nanostructures was 26.33 • due to the partially hydrophilic silica matrix (Figure 2b). After APTMS treatment, the contact angle was slightly increased to 37.38 • (data not shown) because the hydroxyl groups were eliminated by oxane bonding and the less-polar organic silanes were deposited on the surface [43]. The amine groups on APTMS were used to conjugate biomolecules on the surfaces of cavities.

Surface Functionalization of the SiO 2 -IO Nanostructures
To detect the influenza A (H1N1) virus, SiO 2 -IO nanostructures were functionalized as illustrated in Figure 3a. First, the silica surface was functionalized by APTMS to exposure amine groups via siloxane bonding. Then, the IO nanostructure was conjugated with NHS-PEG 4 -maleimide, which is a heterobifunctional cross-linker, to link the amine-functionalized surface to another molecule containing a thiol group. Cys-ProG was previously used for the highly efficient immobilization of the immunoglobulin-binding protein (i.e., IgG) on a gold surface [44]. In the same manner, we conjugated Cys-ProG via the maleimide linker for immobilization of the hemagglutinin (HA) antibody on the IO surface. The surface modification method can provide more stable conjugation sites than the physical absorption method. A previous study reported the detection of targets by electrostatic interactions between the negatively charged, porous TiO 2 surface and the positively charged protein (lysine and/or arginine residues) [27]. In this work, a high concentration of proteins and antibodies (approximately 0.5~2.5 mg/mL) was used in the experiments. To evaluate the surface functionalization of the SiO 2 -IO nanostructures, we measured a series of the reflectance spectra during the modification. As shown in Figure 3b, the reflectance peak was redshifted from 445.41 ± 0.55 nm by approximately 8.05 nm during the APTMS treatment. In addition, the reflectance was further shifted by 3.96 nm and 1.91 nm after treatment with the maleimide linker and Cys-ProG-HA antibody, respectively. The redshifts are results of successful deposition of the molecules on the cavities which slightly increases the refractive index of the medium. We also measured the contact angle to determine the wettability of the solid surface of the opal and SiO2-IO nanostructures. The contact angle is influenced by the surface property of the nanostructures. The contact angle on the opal structure was 82.9° due to hydrophobic colloidal PS beads on the surface of the structure, while that on the SiO2-IO nanostructures was 26.33° due to the partially hydrophilic silica matrix (Figure 2b). After APTMS treatment, the contact angle was slightly increased to 37.38° (data not shown) because the hydroxyl groups were eliminated by oxane bonding and the less-polar organic silanes were deposited on the surface [43]. The amine groups on APTMS were used to conjugate biomolecules on the surfaces of cavities.

Surface Functionalization of the SiO2-IO Nanostructures
To detect the influenza A (H1N1) virus, SiO2-IO nanostructures were functionalized as illustrated in Figure 3a. First, the silica surface was functionalized by APTMS to exposure amine groups via siloxane bonding. Then, the IO nanostructure was conjugated with NHS-PEG4-maleimide, which is a heterobifunctional cross-linker, to link the amine-functionalized surface to another molecule containing a thiol group. Cys-ProG was previously used for the highly efficient immobilization of the immunoglobulin-binding protein (i.e., IgG) on a gold surface [44]. In the same manner, we conjugated Cys-ProG via the maleimide linker for immobilization of the hemagglutinin (HA) antibody on the IO surface. The surface modification method can provide more stable conjugation sites than the physical absorption method. A previous study reported the detection of targets by electrostatic interactions between the negatively charged, porous TiO2 surface and the positively charged protein (lysine and/or arginine residues) [27]. In this work, a high concentration of proteins and antibodies (approximately 0.5~2.5 mg/mL) was used in the experiments. To evaluate the surface functionalization of the SiO2-IO nanostructures, we measured a series of the reflectance spectra during the modification. As shown in Figure 3b, the reflectance peak was redshifted from 445.41 ± 0.55 nm by approximately 8.05 nm during the APTMS treatment. In addition, the reflectance was further shifted by 3.96 nm and 1.91 nm after treatment with the maleimide linker and Cys-ProG-HA antibody, respectively. The redshifts are results of successful deposition of the molecules on the cavities which slightly increases the refractive index of the medium. Antibody immobilization was further confirmed by the HRP activity ( Figure S2). Peroxidase consists of a large family of enzymes and catalyzes the oxidation of the substrate with hydrogen peroxide (H2O2). Peroxidase is widely used in bioanalytical chemistry, for example, to catalyze the conversion of chromogenic substrates, such as 3,3′,5,5′-tetramethylbenzidine (TMB), into colored products [45]. TMB changes to a blue-colored product in the presence of hydrogen peroxide (H2O2) [46]. The surface functionalization was demonstrated by an HRP-tagged antibody. First, we Antibody immobilization was further confirmed by the HRP activity ( Figure S2). Peroxidase consists of a large family of enzymes and catalyzes the oxidation of the substrate with hydrogen peroxide (H 2 O 2 ). Peroxidase is widely used in bioanalytical chemistry, for example, to catalyze the conversion of chromogenic substrates, such as 3,3 ,5,5 -tetramethylbenzidine (TMB), into colored products [45]. TMB changes to a blue-colored product in the presence of hydrogen peroxide (H 2 O 2 ) [46]. The surface functionalization was demonstrated by an HRP-tagged antibody. First, we observed the activity of the functionalized surface. Cys-ProG binds to the antibody through the heavy chains in the region of the Fc fragment. The Cys-ProG-immobilized IO surfaces contain more antibodies than the APTMS-functionalized surfaces. We also compared a SiO 2 thin film with the surface-functionalized inverse opal. Both substrates have hydrophilic hydroxyl groups; however, the IO structure had higher activity (56.5%) than the SiO 2 thin film (19.2%) due to its large surface area.

Detection of the Influenza H1N1 Virus by SiO 2 -IO Nanostructures
We investigated the detection of the pandemic influenza type A (H1N1) virus (A/CA/07/2009) by reflectance measurement. The virus is 80~100 nm in diameter and can be classified into 16 hemagglutinin (HA) subtypes and 9 neuraminidase (NA) subtypes [1]. HA is one of the major surface glycoproteins of the H1N1 subtype and is more prevalent than NA on the viral surface [47]. The viruses were detected by monitoring the immune response between the HA and αHA-1 antibodies. We treated 10 µL of the H1N1 subtype with the antibody-immobilized IO nanostructure by concentration. Figure 4a shows that reflectance peak shift as much as 0.96 ± 0.42 nm, 2.15 ± 0.35 nm, and 2.88 ± 0.36 nm that were measured for the concentration range from 10 3 PFU to 10 5 PFU on the functionalized IO nanostructure. To evaluate the specificity of the functionalized SiO 2 -IO nanostructures, we compared the H1N1 subtype to the H3N2 subtype and IFVB, each with a concentration of 10 4 PFU. SiO 2 -IO nanostructures were prepared by αHA-1 antibody immobilization. The control was treated with PBS buffer solution instead of the virus sample. Figure 4b shows the magnitudes of the redshift of the reflectance peak depending on the type of virus. The H3N2 subtype and IFVB showed a shift that is comparable to the control. Only the H1N1 subtype showed a meaningful magnitude of peak shift, confirming that the antibody-immobilized SiO 2 -IO nanostructures have a high specificity to the H1N1 subtype. observed the activity of the functionalized surface. Cys-ProG binds to the antibody through the heavy chains in the region of the Fc fragment. The Cys-ProG-immobilized IO surfaces contain more antibodies than the APTMS-functionalized surfaces. We also compared a SiO2 thin film with the surface-functionalized inverse opal. Both substrates have hydrophilic hydroxyl groups; however, the IO structure had higher activity (56.5%) than the SiO2 thin film (19.2%) due to its large surface area.

Detection of the Influenza H1N1 Virus by SiO2-IO Nanostructures
We investigated the detection of the pandemic influenza type A (H1N1) virus (A/CA/07/2009) by reflectance measurement. The virus is 80~100 nm in diameter and can be classified into 16 hemagglutinin (HA) subtypes and 9 neuraminidase (NA) subtypes [1]. HA is one of the major surface glycoproteins of the H1N1 subtype and is more prevalent than NA on the viral surface [47]. The viruses were detected by monitoring the immune response between the HA and αHA-1 antibodies. We treated 10 µ L of the H1N1 subtype with the antibody-immobilized IO nanostructure by concentration. Figure 4a shows that reflectance peak shift as much as 0.96 ± 0.42 nm, 2.15 ± 0.35 nm, and 2.88 ± 0.36 nm that were measured for the concentration range from 10 3 PFU to 10 5 PFU on the functionalized IO nanostructure. To evaluate the specificity of the functionalized SiO2-IO nanostructures, we compared the H1N1 subtype to the H3N2 subtype and IFVB, each with a concentration of 10 4 PFU. SiO2-IO nanostructures were prepared by αHA-1 antibody immobilization. The control was treated with PBS buffer solution instead of the virus sample. Figure 4b shows the magnitudes of the redshift of the reflectance peak depending on the type of virus. The H3N2 subtype and IFVB showed a shift that is comparable to the control. Only the H1N1 subtype showed a meaningful magnitude of peak shift, confirming that the antibody-immobilized SiO2-IO nanostructures have a high specificity to the H1N1 subtype. To prove our concept, we used αHA-1 antibody-immobilized AuNPs to visually confirm the virus. Because the GBP-ProG complex can be employed on the AuNP surface, functional linkers can be used for a scanometric antibody probe [48]. The αHA-1 antibody-immobilized AuNPs were treated with the various virus concentrations captured by the SiO2-IO nanostructures. As seen in Figure 5, the SEM images show an increased amount of AuNPs either on the surface or in the pores of the SiO2-IO nanostructures (red arrows in Figure 5d), while there were no AuNPs in the control (Figure 5a). This indicates that virus detection using SiO2-IO nanostructures was indirectly confirmed by the αHA-1 antibody-immobilized AuNPs. To prove our concept, we used αHA-1 antibody-immobilized AuNPs to visually confirm the virus. Because the GBP-ProG complex can be employed on the AuNP surface, functional linkers can be used for a scanometric antibody probe [48]. The αHA-1 antibody-immobilized AuNPs were treated with the various virus concentrations captured by the SiO 2 -IO nanostructures. As seen in Figure 5, the SEM images show an increased amount of AuNPs either on the surface or in the pores of the SiO 2 -IO nanostructures (red arrows in Figure 5d), while there were no AuNPs in the control (Figure 5a). This indicates that virus detection using SiO 2 -IO nanostructures was indirectly confirmed by the αHA-1 antibody-immobilized AuNPs.

Conclusions
In this study, we reported the surface functionalization of SiO2-IO nanostructures with antibodies and their use as biosensors for the detection of influenza viruses. We fabricated the SiO2-IO nanostructures by the colloidal self-assembly method through vertical deposition and calcination processes. For the detection of influenza viruses, we successfully modified the surface of the SiO2-IO nanostructures with antibodies via chemical and biological conjugation. The antibody-immobilized SiO2-IO nanostructure captures the target viruses of the H1N1 subtype when immersed in the dispersion of the viruses, which leads to a redshift of reflectance peak. Therefore, the H1N1 subtype can be simply detected by monitoring the redshift of reflectance peak position in the absence of any labeling procedures. We found that the H1N1 subtype was sensitively and selectively detected in the concentration range from 10 3 to 10 5 PFU. This result was further confirmed by antibody-immobilized AuNPs. The SiO2-IO biosensors have the advantage of large surface area compared with existing optical biosensors due to the three-dimensional nanostructure and enable integration with microfluidics and lab-on-a chip technologies to improve the sensor for point-of-care biosensors. Moreover, various sensing strategies can be employed through the surface functionalization protocol. Therefore, we believe that the SiO2-IO nanostructure-based biosensors can be further utilized for the detection of various biohazards.

Conclusions
In this study, we reported the surface functionalization of SiO 2 -IO nanostructures with antibodies and their use as biosensors for the detection of influenza viruses. We fabricated the SiO 2 -IO nanostructures by the colloidal self-assembly method through vertical deposition and calcination processes. For the detection of influenza viruses, we successfully modified the surface of the SiO 2 -IO nanostructures with antibodies via chemical and biological conjugation. The antibody-immobilized SiO 2 -IO nanostructure captures the target viruses of the H1N1 subtype when immersed in the dispersion of the viruses, which leads to a redshift of reflectance peak. Therefore, the H1N1 subtype can be simply detected by monitoring the redshift of reflectance peak position in the absence of any labeling procedures. We found that the H1N1 subtype was sensitively and selectively detected in the concentration range from 10 3 to 10 5 PFU. This result was further confirmed by antibody-immobilized AuNPs. The SiO 2 -IO biosensors have the advantage of large surface area compared with existing optical biosensors due to the three-dimensional nanostructure and enable integration with microfluidics and lab-on-a chip technologies to improve the sensor for point-of-care biosensors. Moreover, various sensing strategies can be employed through the surface functionalization protocol. Therefore, we believe that the SiO 2 -IO nanostructure-based biosensors can be further utilized for the detection of various biohazards.