Retroreflective Janus Microparticle as a Nonspectroscopic Optical

Apr 15, 2016 - We developed retroreflective Janus microparticles (RJPs) as a novel optical immunosensing probe for use in a nonspectroscopic retrorefl...
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Retroreflective Janus Microparticle as a Nonspectroscopic Optical Immunosensing Probe Yong Duk Han,†,‡ Hyo-Sop Kim,†,‡ Yoo Min Park,†,‡ Hyeong Jin Chun,†,‡ Jae-Ho Kim,†,‡ and Hyun C. Yoon*,†,‡ †

Department of Applied Chemistry & Biological Engineering and ‡Department of Molecular Science & Technology, Ajou University, Suwon 443749, South Korea S Supporting Information *

ABSTRACT: We developed retroreflective Janus microparticles (RJPs) as a novel optical immunosensing probe for use in a nonspectroscopic retroreflection-based immunoassay. By coating the metals on the hemispherical surface of silica particles, highly reflective RJPs were fabricated. On the basis of the retroreflection principle, the RJPs responded to polychromatic white light sources, in contrast to conventional optical probes, which require specific monochromatic light. The retroreflection signals from RJPs were distinctively recognized as shining dots, which can be intuitively counted using a digital camera setup. Using the developed retroreflective immunosensing system, cardiac troponin I, a specific biomarker of acute myocardial infarction, was detected with high sensitivity. On the basis of the demonstrated features of the retroreflective immunosensing platform, we expect that our approach may be applied for various point-of-care-testing applications. KEYWORDS: biosensors, immunoassays, Janus particle, retroreflection, cardiac troponin I

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INTRODUCTION Optical biosensing technology with high accuracy and wide applicability has been intensively investigated for user-centered diagnostic devices in point-of-care-testing (POCT) concept.1−4 Although numerous studies have been conducted, the commercialization of POCT optical biosensors has been limited because current approaches employ sophisticated spectrometric optics components (e.g., monochromator with halogen lamp, laser, optical filter, and spectrometer) for detecting optical signals of the employed optical probes.5−7 The traditionally used optical signaling probes (e.g., enzymes, chromogens, nanoparticles, and fluorophores) generate varied spectroscopic signals such as color formation, spectral shifting, or fluorescence, which can be analyzed using only wavelengthselective optics components exhibiting limitations in commercialization and miniaturization such as excessive cost, high power consumption, and complicated configuration.8−12 Thus, for the materialization of simple POCT optical biosensor, the introduction of a novel optical probe whose signal can be analyzed using a nonspectroscopic instrument, thereby enabling easy detection, is strongly desired.13,14 To address this, we employed the retroreflection principle and retroreflectors, which are currently applied in various fields such as safety clothing, road signs, vehicles, and optical communications, for optical biosensing signal registration.15−21 Retroreflection is a unique phenomenon of light irradiating a surface and being redirected back to the light source (Figure 1). The material and its surface, inducing the retroreflection, are called a retroreflector. Interestingly, the retroreflector responds to © 2016 American Chemical Society

Figure 1. Schematic illustration of a typical light path in retroreflection. When the light is irradiated on the retroreflector surface, the light is reflected back to the direction of light source and observer. Inset shows the typical light path in specular reflection. In the specular reflection, the reflected light cannot be observed when the observer is located in the same direction with light source.

various light sources, including visible light, infrared light, and even nonmonochromatic light.16,19,20 Thus, retroreflectorassociated retroreflection can be easily induced and detected Received: February 19, 2016 Accepted: April 15, 2016 Published: April 15, 2016 10767

DOI: 10.1021/acsami.6b02014 ACS Appl. Mater. Interfaces 2016, 8, 10767−10774

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incident light reaches the rear surface of a silica particle, this region of the particle acts as a concave spherical mirror with the required curvature for retroreflection; the light is internally reflected back through the frontal surface of particle and subsequently refracted in a direction parallel to its origin (Figure 4a).15−18 However, at the rear surface of the particle, a significant portion of incident light passes through rather than being internally reflected because of its transparent property (Figure 4b). To prevent this and improve retroreflectivity, highly reflective metal layers were coated on the hemispherical surface of silica microparticles. Using the Langmuir−Blodgett (LB) film technique and the electron beam physical vapor deposition (EBPVD), 40 nm aluminum and 20 nm gold layers were sequentially constructed on the hemispherical surface of the silica particles (Scheme 1).29,30 The aluminum layer exhibiting high reflectance was chosen as a retroreflectivity enhancer and as an intermediate adhesive layer for the deposition of gold, which adheres poorly to the silica surface. The gold layer was employed to enhance the spatio-selective functionalization of the reacting antibodies using the selfassembled monolayer (SAM) technique. Figure 2a,b shows images of the resulting RJPs which are closely packed on the glass substrate after EBPVD. Scanning electron microscopy (SEM) images of the isolated RJP (Figure 2c) show that the metal layer is deposited only on the hemispherical region of the particle. Additionally, the elemental analysis results from energy-dispersive X-ray spectroscopy supported the idea that each metal was successfully deposited onto the particle (Figure S1 and Supplementary Methods S2). Quantitative Analysis of the Retroreflective Property of RJPs. To investigate whether RJPs can be used as retroreflector, its retroreflective properties were analyzed. Given the asymmetric structure of RJPs, the transparent silica surface of RJPs should be exposed toward the direction of the light source. Thus, for proper sample preparation, RJPs with 40 nm Al layer and 20 nm Au layer were transferred from the glass substrate to a carbon tape (Figure 5a). As a control, a carbon tape which was covered with nontreated silica particles was also prepared and evaluated. The SEM image in Figure 5a (right panel) shows that the RJPs were attached to the tape in the desired orientations. The quantitative evaluation of the retroreflection from RJPs was carried out using a homemade retroreflection analysis system, enabling the selective separation of retroreflected light from the incident and specularly reflected lights (Figures 5b and S2a and Supplementary Methods S3). Three different diode lasers and a white LED were utilized as light sources. The retroreflection intensities from the RJP sample were enhanced using 405, 532, and 655 nm laser sources by 450, 240, and 180%, respectively, compared with those of the nontreated silica particles (Figure 5c). These changes in the intensity of retroreflection according to the types of sample could be traced by direct observation (Figure S2b− e). As presented in Figure S2c−e, the retroreflected red laser light from RJPs was brighter than the light reflected from carbon tape and silica particle. Experiments using white LED as a light source exhibited similar results (Figure 5d). Over the entire LED spectrum, stronger retroreflection was observed with the RJPs than with the silica particles. On the basis of the developed retroreflection analysis system, the effect of aluminum thickness in RJPs to the retroreflection property was evaluated (see details in Supplementary Methods S4). Prior to the test, three types of RJPs exhibiting different thicknesses (20, 40, and 80 nm) of aluminum layer were

via a nonspectroscopic analytical approach. On the basis of this feature, we developed retroreflective Janus microparticles (RJPs) as a new optical immunosensing probe (Figure 2).22−24 RJPs were designed to provide bright retroreflection

Figure 2. (a) Photograph of the fabricated RJPs on the glass substrate after the metal deposition process. The close-packed arrangement of thin gold film-coated RJPs induces structural diffraction and generates rainbow-like colors. (b) SEM image of the fabricated layer of closely packed RJPs on the glass substrate. (c) SEM image of the isolated single RJP.

signals in the sandwich immunoassay; these signals can be easily detected using polychromatic white light-emitting diode (LED) irradiation. To demonstrate the applicability of RJPs as immunosensing probes, we tested cardiac troponin I (cTnI), an acute myocardial infarction (AMI)-specific biomarker.25−27 The retroreflection signals from RJPs in cTnI immunosensing appear as shining dots, similar to luminous retroreflective road signs visible at night, which can be intuitively recognized and easily counted (Figure 3).

Figure 3. (a) Schematic diagram for the strategy of the proposed retroreflective cTnI sandwich immunoassay using RJP as an optical immunosensing probe. (b) Retroreflective road signs at night time. When the headlight of automobile irradiates the sign, the road signs are brightly shown via retroreflection. (c) Schematic illustrations and figures of retroreflective immunosensing surface before (left) and after (right) the white LED illumination. Similar to the retroreflective road signs, the RJPs are brightly shown via retroreflection.

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RESULTS AND DISCUSSION Design and Fabrication of Retroreflective Janus Particles. Few studies have attempted to utilize retroreflectors as optical probes because they can only be fabricated by a sophisticated photolithographic process, limiting the commercialization of the probes.21 Considering the facile fabrication, RJPs were prepared with a simple structure of a transparent ball lens retroreflector. In this study, transparent silica microparticles (SiO2, diameter ≈ 1.2 μm) synthesized using the Stöber method were chosen as a ball lens material.28 When 10768

DOI: 10.1021/acsami.6b02014 ACS Appl. Mater. Interfaces 2016, 8, 10767−10774

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Figure 4. Schematic illustration of light paths in the utilized microparticles (a) Light paths in the developed RJP. (b) Light paths in the transparent silica microparticle.

Scheme 1. RJP Fabrication Process Utilizing the LB Technique and EBPVD

Figure 5. (a) Schematic illustration of sample preparation procedure for RJP retroreflection analysis. Right panel shows SEM image of RJPs after transferring from a glass substrate to an adhesive carbon tape. By the transferring process, silica region of RJPs was exposed to the direction of light source. (b) Illustration of the developed retroreflection observation setup. (c) Spectral analysis of the retroreflected light from RJPs and silica particles using diode laser as a light source (405, 532, and 655 nm). Carbon tape was used as a background sample. (d) Spectral analysis of retroreflected light from RJPs and silica particles using white LED.

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ACS Applied Materials & Interfaces prepared (thickness of gold layer was fixed at 20 nm). The prepared RJPs were transferred to carbon tape and their retroreflection properties were quantitatively analyzed using 405 nm laser source. As depicted in Figure 6a, the

substrate, ultrasonication was conducted. However, a too thick metal layer of RJPs reduces the RJP dispersion. As shown in Figure 6b, the RJPs with 80 nm aluminum layer were not separated into isolated single particles and remained as several clusters after the ultrasonication (for 30 s). In contrast, the RJPs with 40 nm aluminum layer were easily separated into isolated single particles via ultrasonication as depicted in Figure 6c. Considering the retroreflection yield and separation efficiency, the RJPs with 40 nm aluminum and 20 nm gold layers were used in the following study. Qualitative Evaluation of the Retroreflective Property of RJPs. For the qualitative evaluation of retroreflection from the particles, a letter-patterned mask comprised of a transparent letter pattern and black wax-covered region was mounted on top of the particle-displayed samples (Figure 7a). Qualitative

Figure 7. (a) Schematic illustration of sample preparation procedure for qualitative RJP retroreflection analysis. The letter-patterned mask was mounted on the particle-displayed carbon tape. (b) Qualitative evaluation result of retroreflection from RJPs (left) and nontreated silica microparticles (right).

analysis showed that RJP- and silica-microparticle-associated samples were visually similar under indoor light conditions (Figure 7b, upper panels). When the LED flashlight was applied, however, the letter pattern of RJPs was distinct, whereas the same pattern for the silica particles disappeared (Figure 7b, lower panels). For silica particles, the applied light penetrated the rear surface of the particle and showed very weak retroreflection. In comparison, for the RJPs-associated surface, light penetration was prevented by the Al/Au metal layers on the hemispherical surface of RJPs. Thus, the retroreflection of RJPs was much more intense than that of the silica particles. On the basis of the obtained retroreflection analysis, we concluded that the developed RJPs can be used as a retroreflector under diverse light source conditions. Characterization of Optical Signal from RJPs under the Microscopic Imaging System. To investigate further how the retroreflection signals from RJPs behave in a microscopic imaging system, the RJP sample was dispersed onto a microscopic slide and analyzed under a bright field microscope with an external white LED light source (Figure S3). Figure 8a and Movie S1 show that the retroreflected light from RJPs appeared as bright white dots in the designed imaging system. Interestingly, free RJPs, which were floating in the buffer solution, blinked continuously (Figure 8b). This blinking of RJPs can be explained by the RJPs freely rotating in the liquid phase. When the RJP rotates in buffer solution, its metal layer and transparent silica region are alternately exposed to the incoming light source, resulting in the turning on and off of the retroreflected light. In contrast, the transparent nontreated silica particles appeared as pale white dots under

Figure 6. (a) Spectra of the retroreflected laser light (405 nm) from three types of RJPs with varied aluminum thicknesses (20, 40, and 80 nm). The thickness of gold layer was fixed at 20 nm. Carbon tape was used as a background sample. (b) Bright-field microscopic image of the buffer-dispersed RJPs with 80 nm of aluminum thickness. The RJPs were not isolated and remained as clusters. (c) Bright-field microscopic image of the buffer-dispersed RJPs with 40 nm of aluminum thickness. The RJPs were isolated as single particles and dispersed in buffer solution.

retroreflection signals from each RJP were proportional to the thickness of aluminum layer on the RJP. This result indicates that the thicker reflective layer (aluminum) is suitable for the generation of intensive retroreflection. However, for the practical use of RJPs as an optical probe in the sandwich immunoassay, the thickness of metal layers on the RJP should be considered from the viewpoint of particle isolation and dispersion. In the RJP fabrication, the RJPs are closely packed on the glass substrate after the LB-filming and metal deposition procedures. Each close-packed RJP on the glass substrate is connected to each other by the deposited metal layers. To isolate and disperse the interconnected RJPs from the glass 10770

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Scheme 2. Schematic Illustration of the Biofunctionalization Process for RJPsa

Figure 8. (a) Microscopic image of RJPs dispersed in PBS. Shining dots are RJPs. (b) Magnified images and schematic illustrations of single RJP, blinking by the free rotation in solution. (c) Microscopic image of silica microparticles, dispersed in PBS. The pale white dots are unmodified silica microparticles.

the microscope, and their intensities were maintained for an extended amount of time (Figure 8c). This distinctive difference in retroreflection between the RJPs and silica particles could be expressed by the Fresnel equation (eq 1).17,18 Under normal light incidence (incident angle = 0°) from medium A into the interfacial material B, the % reflectivity of the interfacial layer (R) can be calculated as follows: R (%) = [(nA − nB)/(nB + nA )]2 × 100

(1)

where nA and nB are the refractive indices of the medium A and interfacial material B, respectively. Using this equation, the reflectivity of RJP was found to be 11.20% for 532 nm light, which was enhanced by up to 40-fold from the calculated reflectivity of silica particles (0.21%, Tables S1−S4).31−33 The aluminum layer on the surface of RJPs appeared to play a critical role in retroreflection. On the basis of this result, we confirmed that the newly designed RJPs could be utilized as retroreflectors under microscopic imaging system with nonmonochromatic light conditions. Spatio-Selective Biofunctionalization of RJP. The microscopic test results demonstrated that the right orientation of the RJPs toward the incident light was essential for retroreflective signal generation. Therefore, a spatio-selective antibody functionalization on RJPs is required. We addressed this issue by introducing a gold surface-specific SAM technique and UV-initiated photo-cross-linking method (Scheme 2). On the gold surface of RJPs, mouse IgG antibodies were spatio-selectively modified (SMJPs) by using amine-reactive SAM (3,3′-dithiobis[sulfosuccinimidylpropionate], DTSSP), polyamidoamine dendrimer (Dend) containing 64 primary amine groups, and photoreactive cross-linker (sulfo-NHS-LCdiazirine, SDA) as shown in Scheme 2a (see details in the Experimental Section). As a positive control, RJPs were randomly conjugated with mouse IgG using only DTSSP SAM (RMJPs, Scheme 2b). As a negative control, bovine serum albumin (BSA)-modified RJPs (BMJPs) were prepared in the same manner as RMJPs. To validate the antibody modifications in SMJPs and RMJPs, a fluorescence microscopic study was conducted using Alexa Fluor488-conjugated anti-mouse IgG (Figure 9; see details in Supplementary Methods S5). For BMJPs, no fluorescence was observed (negative control, Figure 9a), indicating that all metal-coated and silica-exposed

a

(a) Procedure of spatio-selective antibody conjugation to the RJPs (SMJPs). (b) Procedure of random antibody conjugation to the RJPs (RMJPs).

Figure 9. Fluorescence microscope images of mouse IgG-immobilized particles including (a) BMJP, (b) RMJP, and (c) SMJP after the labeling with AlexaFluor488-conjugated anti-mouse IgG.

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ACS Applied Materials & Interfaces hemispherical surfaces on the RJPs were completely blocked by BSA. In contrast to BMJPs, strong green fluorescence appeared on the entire surface of RMJPs (positive control, Figure 9b). As predicted, the mouse IgG antibody was randomly modified on the RMJPs through covalent bonding and physical adsorption. For SMJPs, however, fluorescence signals appeared only on the hemispherical surface of the particle where the Al/Au metal layer was deposited (Figure 9c). These results suggest that the spatio-selective antibody functionalization of SMJPs was achieved using the employed photo-cross-linking method. Retroreflection-Based cTnI Immunosensing. To verify the applicability of RJPs as optical labels for the immunosensing of disease markers, a retroreflective sandwich immunoassay using SMJP for cTnI, an AMI-specific biomarker, was conducted.25−27 For the accurate and facile retroreflectionbased cTnI immunoanalysis, the RJP-quantifying chip (RQC) containing 16 immunosensing zones was fabricated and used (Figure S4a−c; see details in Supplementary Methods S6, Supporting Information). On the immunosensing zones comprised of gold thin-film (200 nm), the cTnI immunosensing surfaces were prepared by the immobilization of cTnI capturing antibodies (Figure S4d). To the prepared cTnI immunosensing surface on the RQC, various concentrations of cTnI samples (0−100 ng/mL) and cTnI signaling antibodyconjugated SMJP probes (0.4 mg/mL) were sequentially applied (Figure S4d). Then, the images of resulting cTnI immunosensing surfaces were observed and digitally registered using a digital imaging setup which is comprised of an external white LED light source, a complementary metal oxide semiconductor (CMOS) digital camera and a zoom lens (Figure S5). As a result, the number of shining dots in the reaction zone (the square-patterned gold surface, 340 × 340 μm2 in dimension) was increased in proportion to cTnI concentration (Figure 10a). Assuming the shining dots indicate the RJPs retroreflecting white LED light (Figure 8), the increase in number of shining dots on the gold surface could be interpreted as cTnI antibody-conjugated RJP probes which were bound to the immunosensing zone via sandwich immunoaffinity reaction to the target analytes (cTnI). By counting the number of shining RJPs in the sensing zone using NIH ImageJ software, a linear dose−response curve for cTnI was obtained (Figure 10b).25 The calibration result yielded a cTnI detection range from 0.01 to 100 ng/mL. The limit of detection for cTnI immunosensing, calculated as 3.3 times the standard deviation of the background signal divided by the slope of the calibration curve based on the ICH guidelines, was as low as 0.05 ng/mL.34 Because the conventional cutoff level of cTnI for AMI diagnosis is lower than 0.1 ng/mL, the analytical performance of the developed RJP label and the retroreflection-based immunosensing system fulfill the clinical requirements for cTnI detection.25−27 Noticeable features of the developed probe and its immunosensing application are the use of extremely simplified nonspectroscopic optics system including a polychromatic white LED light source and a CMOS digital camera with low-magnification zoom lens exhibiting a 0.065 numerical aperture (NA). With regard to the requirements of highmagnification objective lens and fluorescence filter unit for conventional biomarker detection utilizing fluorophores or fluorescence microbeads as signaling probes, these changes are significant advances that simplify the optical immunosensing system and enable its integration into IT devices such as smartphones.25,35,36

Figure 10. (a) Results of retroreflective cTnI immunoassay utilizing cTnI antibody-conjugated SMJPs. (b) Dose−response curve for number of SMJPs in the square-patterned gold immunosensing zone (340 × 340 μm2 in dimension) as a function of cTnI concentration. Each data point represents the average and standard deviation of independent triplicate tests.

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CONCLUSIONS We developed a novel retroreflective optical immunosensing probe. The probe, RJP, can be used for an intuitive and accurate retroreflection-based nonspectroscopic immunoassay employing minimal optics setup including general digital camera and polychromatic LED flash. On the basis of the features of our retroreflective sensing platform, we expect that this approach can be applied for various bioanalytical applications under the concept of POCT.

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EXPERIMENTAL SECTION

Synthesis of Monodispersed Silica Microparticles. Monodispersed silica particles (1.2 μm in diameter) were prepared by the Stöber method utilizing the hydrolysis and condensation of tetraethyl orthosilicate (TEOS). Prior to synthesis, a catalyst solution was prepared by mixing 1 L of ethanol (EtOH), 40 mL of ammonia, and 80 mL of double-distilled and deionized water (DDW). Under vigorous stirring, 15 mL of TEOS was injected into 300 mL of catalyst solution to synthesize 300 nm diameter seed. Next, to synthesize monodispersed silica particles 1.2 μm in diameter, the collected seed particles were dispersed in the mixture solution containing 600 mL of the catalyst solution containing 2 μM potassium chloride under vigorous stirring. Subsequently, 40 mL of TEOS was added dropwise to the mixture for 20 h. The synthesized monodispersed silica microparticles were recollected by centrifugation (4000 rpm for 10 min) and dispersed in EtOH. These procedures were repeated three 10772

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ACS Applied Materials & Interfaces times. Finally, the silica particles were dried in an air-circulating oven at 110 °C for 24 h. Fabrication of RJPs. To construct an LB film of silica particles on water, the silica particles should be dispersed along the air−water interface. However, the synthesized silica particles are too hydrophilic to be dispersed along the water surface. To overcome this, the surface of silica particle was modified by carbodiimidazole (CDI) to increase hydrophobicity. Prior to surface modification, synthesized silica microparticles (1.2 μm in diameter; 2 g) were dispersed in 40 mL of EtOH. Next, 1 g of CDI was added to the mixture and allowed to react for 1 h under vigorous stirring. The resulting precipitated particles were dispersed by sonication and were repetitively washed to remove the unreacted CDI. Then, the CDI-treated silica particles were dried in a vacuum oven for 24 h. The CDI-modified silica particles dispersed in chloroform (2 mg/ mL) were compressed into a hexagonally close-packed monolayer film at the air/water interface of a Langmuir trough and transferred to a coverslip for a microscope slide (18 × 18 mm2, BK7 glass) by lifting at a constant speed (1 mm/min). Under this setting, at least 34 separate template films could be prepared simultaneously. Subsequently, a 40 nm thick aluminum (Al) layer was deposited onto the silica particleassociated LB film using an e-beam physical vapor deposition (EBPVD). After aluminum deposition, a 20 nm-thick gold (Au) layer was deposited onto the silica/Al-associated LB film on the glass substrate using the EBPVD. The RJP fabrication process is schematically illustrated in Scheme 1. Spatio-Selective Antibody Conjugation on RJPs. To induce an effective retroreflection signal from the RJP, which was utilized as an optical immunosensing probe, a strategy for spatio-selective antibody conjugation on RJPs is required. To address this, we employed selfassembled monolayer (SAM) technology, a dendrimer and photocross-linking method using SDA (Scheme 2a). First, an amine-reactive SAM was constructed onto the gold-coated hemispherical surface of RJPs on a glass substrate by dipping the sample into 5 mM DTSSP solution for 3 h. After rinsing with DDW, 0.5% Dend (in DDW) was applied to the DTSSP-modified RJPs and allowed to react for 1 h. During the process, dendrimers were covalently bound to the DTSSP SAM-modified gold region via amide bond formation between the amine group of dendrimer and the sulfosuccinimidyl ester (sNHS) of DTSSP. Because the utilized dendrimer is spherical polymer containing 64 primary amine groups, the dendrimer-treatment yields an amine-rich surface on the gold layer of RJPs. To block the unreacted sNHS moieties of the DTSSP-modified gold surface, the sample was treated with 10 mM ethanolamine (EA) for 30 min. Next, 20 mM SDA, a bifunctional cross-linker containing two types of amine reactive moieties, including a photoreactive diazirine group and sNHS, was loaded onto the Dend-modified surface and allowed to react under darkroom conditions for 30 min. Through amide bond formation between the sNHS moieties of SDA and the amine groups of Dend, SDA was functionalized on the modified RJPs. To passivate the silicaexposed hemispherical surface of RJPs, 0.2% BSA was applied and allowed to physically adsorb onto the silica surface of RJPs under dark conditions for 1 h. In this procedure, BSA would be nonspecifically bound to the hemispherical silica surface of RJPs. Because the diazirine moiety of dendrimer-conjugated SDA requires UV light to be conjugated with primary amine in the lysine side chain of protein molecules, BSA on the gold layer would not be immobilized to SDAterminated gold surface. After washing with PBS, 150 μg/mL mouse IgG antibody was reacted with the SDA-modified RJP-associated substrate. Next, UV light (365 nm) was irradiated for 30 min to conjugate the antibody to the diazirine groups on RJPs. By the UV light, the primary amines in the lysine side chain of antibody were covalently conjugated with diazirine functional groups of SDA on the gold layer of RJPs. And then 0.2% BSA was applied to the RJPs and UV light was irradiated for 10 min to passivate the unreacted diazirine moieties. Finally, the resulting RJPs having an antibody-functionalized hemisphere, the spatio-selectively modified RJPs (SMJPs), were isolated from the substrate by ultrasonication and transferred into PBSB buffer.

As positive and negative controls, randomly modified RJPs (RMJPs) and BSA-modified RJPs (BMJPs) were also prepared. For RMJP preparation, 5 mM DTSSP was applied to the RJPs on the glass substrate for 3 h to construct an amine-reactive SAM (Scheme 2b). After rinsing, 150 μg/mL mouse IgG was directly applied to the DTSSP-modified RJPs without the treatment of dendrimers and SDA. In this step, the antibodies would be nonspecifically bound to the hemispherical silica surface of RJPs via physical adsorption, whereas the antibodies on the gold layer would be conjugated to the sulfo-NHS moiety of DTSSP on gold layer of RJP via amide bond formation. After rinsing with PBS, unreacted sNHS moieties were blocked by treatment with 10 mM EA and 0.2% BSA. The resulting RMJPs were transferred to PBSB buffer by ultrasonication. As a negative control, BMJPs were prepared using a similar method as was used for RMJPs. In the protein conjugation step for BMJP, BSA was applied to the DTSSP-modified RJPs, instead of mouse IgG for RMJPs. Preparation of the Immunosensing Surface and RJP Probe for cTnI Immunoassay. For the construction of the cTnI immunosensing surface on the RJP-quantifying chip (RQC), the chip surfaces were cleaned by immersing them in a piranha solution for 5 min. (Caution! Piranha solution reacts violently with most organic materials and must be handled with extreme care.) After cleaning, 5 mM DTSSP in DDW was applied to the gold chip surface and allowed to react for 3 h. After rinsing with DDW, 150 μg/mL of cTnI capturing antibody (625 clone) was loaded and reacted with DTSSP for 1 h (Figure S4d). Next, using PBS, the antibody-modified gold surfaces were rinsed to remove unreacted antibodies. Next, to block unreacted sNHS on the surface, 10 mM EA and 0.2% BSA were applied for 30 min. The resulting cTnI antibody-immobilized RQCs were stored in PBSB at 4 °C. To prepare spatio-selectively modified RJP (SMJP) immunosensing probes for target biomarker (cTnI) detection, cTnI signaling antibody (19C7 clone) was introduced and spatio-selectively conjugated with RJPs. Retroreflective Immunosensing of cTnI. Retroreflective sandwich type cTnI immunosensing was accomplished as described below. Prior to immunosensing, various concentrations of cTnI samples were prepared in PBS (0, 0.01, 0.05, 0.1, 1, 10, and 100 ng/ mL). A cTnI sample was applied to the capturing antibody-modified cTnI immunosensing surface of RQC and allowed to react for 30 min. After rinsing with PBS, 0.4 mg/mL of the cTnI signaling antibodyconjugated SMJPs were applied for 25 min (Figure S4d). After incubation, the resulting surface was rinsed and analyzed using a newly developed imaging setup described below. Retroreflective Immunoanalysis on an Imaging Setup. For the intuitive retroreflection-based optical immunoanalysis of cTnI, a digital imaging setup was constructed (Figure S5). A CMOS digital camera with 0.065 numerical aperture zoom lens (magnification range = ×0.75−4.5) was used. As the light source, a white LED was employed. The imaging device and LED light source were installed vertically above the immunosensing surface. The incidence angle of LED light toward the sample was 45° to selectively register the retroreflection images from the immunosensing surface with minimized signal interference from specular reflection. The CMOS camera was connected to a laptop computer, and the acquired images were monitored in real-time. During the illumination of the white LED to the surface-bound RJPs, the cTnI immunosensing surfaces were digitally documented as image files. The number of shining dots in the reaction zone (340 × 340 μm2 in dimension) were quantified using the NIH ImageJ software.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b02014. Methods (section S1−S6), supporting figures (Figures S1−S5), supporting tables (Tables S1−S4), and references (S1−S5). (PDF) Movie S1 showing retroreflective Janus particles. (AVI) 10773

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by Samsung Research Funding Center of Samsung Electronics under Project Number SRFCIT1401-02.

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DOI: 10.1021/acsami.6b02014 ACS Appl. Mater. Interfaces 2016, 8, 10767−10774