Cholesteric Liquid Crystal Droplets for Biosensors - ACS Applied

Sep 12, 2016 - By utilizing a microfluidics approach, we prepared uniformly sized cholesteric liquid crystal (CLC) droplets from MLC-2132 doped with a...
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Cholesteric Liquid Crystal Droplets for Biosensors Hyun-Gyu Lee, Sundas Munir, and Soo-Young Park* Department of Polymer Science & Engineering, Polymeric Nanomaterials Laboratory, School of Applied Chemical Engineering, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 41566, Korea S Supporting Information *

ABSTRACT: By utilizing a microfluidics approach, we prepared uniformly sized cholesteric liquid crystal (CLC) droplets from MLC-2132 doped with a chiral dopant (S)-4-cyano-4′-(2-methylbutyl)biphenyl (CB15). We studied the helical structures and reflecting color patterns of high- and low-dopant CLC droplets coated with poly(vinyl alcohol) (PVA) and sodium dodecyl sulfate (SDS). One central large spot with reflecting color in the CLC droplets (initially coated with PVA for planar anchoring) changed to many small spots with the same reflecting color (chicken-skin pattern) when an SDS aqueous solution was introduced to increase the homeotropic anchoring power. These small spots subsequently merged into several spots (flashlight pattern) with time. The CLC droplets coated with poly(acrylic acid)-b-poly(4-cyanobiphenyl-4′-oxyundecyl acrylate) (PAA-bLCP) (CLCPAA droplets) were pH-responsive. Their helical structure and the reflecting color pattern changed because of protonation (at low pH) and deprotonation (at high pH) of the carboxylic group of PAA, which causes the planar (tangential) and perpendicular (homeotropic) orientations, respectively. The CLCPAA droplets immobilized with glucose oxidase (GOx) and cholesterol oxidase (ChO) (CLCPAA‑GOx and CLCPAA‑ChO droplets, respectively), for glucose and cholesterol detection, exhibited high sensitivity (0.5 and 2.5 μM for the CLCPAA‑GOx and CLCPAA‑ChO droplets, respectively), good selectivity, and fast response (≤4 s). Further optimization will enhance their performance as biosensors. With this novel approach, detection is possible by observing the coloring pattern of CLC droplets, without the crossed polarizers that are necessary for nematic LC biosensor systems. KEYWORDS: cholesteric liquid crystal, droplet, microfluidics, enzyme, biosensor

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an enzymatic reaction).13,14,24−31 This LC-based reporting via orientational change offers potential advantages over conventional techniques because it does not require complex instrumentation or labeling. Most previous research has focused on using nematic LC (NLC) to sense biomaterials at LC interfaces. Another important type of LC, the cholesteric liquid crystal (CLC), has been widely used in electro-optic materials in display devices and in other responsive materials. Beside linear anisotropy and birefringence (as in nematic LCs), chirality at the supramolecular level is present in the cholesteric phase, where the helical configuration of the molecular director is responsible for interesting properties, such as selective reflection. CLCs have the ability to reflect circular polarized light at a given wavelength because of the self-organizing

INTRODUCTION Liquid crystalline materials are promising candidates for the sensing of biomaterials.1−12 Liquid crystal (LC) ordering is highly sensitive to molecular-level physical and chemical interactions at the LC interface, which lead to changes in optical appearance. This property allows events that occur at the nanoscale level to be observed using optical microscopy. Furthermore, it is possible to tailor LC-aqueous interfaces in ways that lead to changes in the orientational order of the LC upon external stimuli. For example, the orientational ordering transition of thin LC films in a transmission electron microscopy (TEM) grid can be triggered by enzymatic reactions and interactions with lipids,13−15 surfactants,1,6,16,17 proteins,18−20 DNA,3,21,22 and viruses10,23 at the modified LCaqueous interface. The nature and extent of these changes is influenced by the structure of the modifying material at the LC interface (e.g., surfactant tail length or headgroup structure)18,20 and the types of processes at the LC interface that disrupt or perturb the LC orientation (e.g., local pH change by © 2016 American Chemical Society

Received: August 2, 2016 Accepted: September 12, 2016 Published: September 12, 2016 26407

DOI: 10.1021/acsami.6b09624 ACS Appl. Mater. Interfaces 2016, 8, 26407−26417

Research Article

ACS Applied Materials & Interfaces

thereby according wider applications to advanced photonic devices. If the tangential anchoring on the surface of a CLC droplet could be changed to homeotropic (perpendicular) anchoring by external stimuli, then CLC droplets could be used as LC biosensors. To produce stimuli-responsive CLC droplets with a shape-persistent dopant, the surfaces of the CLC droplets should be functionalized with stimuli-responsive materials. Microfluidic methods can produce monodispersed functionalized NLC droplets for biosensing by coating stimuliresponsive materials. Environmental changes such as pH variation, protein binding, and enzymatic catalysis can cause the functional groups on these NLC droplets to induce the anchoring orientation to change between parallel and perpendicular13,14,53,54 The coating materials can be surfactants,18 polyelectrolytes,31 and pH-sensitive block copolymers.30,55 NLC droplets with parallel (planar; P) and perpendicular (homeotropic; H) anchoring configurations are called bipolar and radial droplets, respectively. Bipolar and radial NLC droplets are typically produced by coating their surfaces with poly(vinyl alcohol) (PVA) and sodium dodecyl sulfate (SDS), respectively.56 NLC droplets can also be functionalized with block copolymers of polyelectrolyte-bSGLCPs (SGLCP: side group LC polymer), where the polyelectrolyte block is in a charged state and the SGLCP block anchors the LC at the LC-aqueous interface.30 Strong polyelectrolytes such as poly(sodium styrenesulfonate) (PSS) and quaternized poly(4-vinylpyridine) (QP4VP) induce the H configuration of NLC droplets regardless of pH; however, weak polyelectrolytes such as poly[(dimethylamino)ethyl methacrylate] (PDMAEDA) and poly(acrylic acid) (PAA) induce an H or P configuration of NLC droplets depending on the pH.27,28,52,57,58 Poly(acrylic acid)-b-poly(4-cyanobiphenyl-4′oxyundecyl acrylate) (PAA-b-LCP) is a coating material that enables NLC droplets to change their configuration via protonation and deprotonation of the PAA carboxylic group.52 At the LC-aqueous interface, the charged state of high-pH PAA induces the H configuration through deprotonation, and the neutral state of low-pH PAA induces the P configuration through protonation. A convenient feature of PAA-b-LCP as a droplet-coating material is that it can couple to amino groups via an N-hydroxysuccinimide/1-ethyl-3-(3(dimethylamino)propyl)carbodiimide (EDC/NHS) reaction. Several enzymes such as glucose oxidase (GOx), cholesterol oxidase (ChO), and urease have been coupled to the surfaces of PAA-decorated NLC droplets to detect glucose,14 cholesterol,15 and urea,13 respectively. Combining the versatility of PAA-bLCP with the unique photonic structure of cholesteric LC droplets may enable biosensing without polarized light. In the present study, we produced uniformly sized CLC droplets, and functionalized them with PAA-b-LCP (denoted CLCPAA droplets), using a microfluidics method. We observed the effects of the charge state of the CLC surface on the configuration of the CLCPAA droplets. A surface electric field, generated by the charge state of a polyelectrolyte, exerts a poorly understood influence on the internal bulk orientation of a decorated CLC droplet. To investigate this influence and its implications for practical biosensor development, we immobilized the enzymes glucose oxidase (GOx) and cholesterol oxidase (ChO) on CLCPAA droplets with high or low chiral dopant content, and observed the changes that occurred in their coloring patterns and helical structures in the presence of glucose or cholesterol.

molecular helices. These properties have also proven to be effective for constructing optical sensors. In these sensors, a responsive molecular trigger provides specificity that leads to an amplified material response, resulting in a change of the reflection band. The reflection band of a CLC can be tuned by stimuli, such as temperature and light.32−43 In comparison to an NLC, a CLC has additional helical organization along the orientation of the director. This organization is characterized by a helical pitch (p), which is the distance corresponding to a 360° rotation of the mesogens along the helix director. If the length of the pitch is of the same order of magnitude as the wavelength of visible light, then the cholesteric phase selectively reflects light and shows a color in the visible range. Moreover, the reflecting colors of CLC are dependent on the pitch length. The wavelength of the selective reflection can be defined as λ = np, where n is the mean refractive index of the LC.44,45 If the pitch p changes in response to environmental variations in the visible range, and consequently, color (or structure) changes are observable, then a CLC can be used as an optical detector of external stimuli. The CLC phase can occur either when the mesogen is chiral or when the director is twisted by doping NLC with a chiral dopant. The helical structure of the CLC is originated by the chirality of the LC molecules. However, an achiral nematic LC can form the helical structure by doping with a chiral dopant and the achiral LC doped with a non-LC chiral moiety can exhibit the helical structure. The helical structure itself is not necessary to be chiral. The chirality does not affect the reflecting color patterns which is only dependent on the amount of chiral dopant. The helical twisting power (HTP) of a dopant is defined as HTP = 1/(pc), where c is the concentration of the dopant. Doped CLC have many favorable properties compared to CLCs, consisting of chiral mesogens. One of the advantages of a doped CLC is the ease with which its pitch can be tuned by changing the contents of the dopant46 Chiral dopants can be shape-persistent or -switchable in response to environmental changes. Photo- or heat-responsive sensors have been implemented using shape-switchable chiral dopants.36,37,42 However, synthesis of shape-switchable chiral dopants is sometimes difficult and time-consuming. Lee et al. have reported a microfluidic-based approach for the controlled formation of photonic microcapsules. They made the CLC droplet with the elastomeric solid membrane using tripleemulsion drops as the template to maintain their planar alignment. However, they did not functionalize the surface of the microcapsule for biosensor applications.47 LC-based sensing platforms include transmission electron microscopy (TEM) grids,1−12,15−17 glass capillaries,48 and droplets.13,14 Droplets have attracted much attention because the geometric confinements of the LC in micrometer-sized droplets exhibit varied configurations, which depend on how the LC orientation (e.g., parallel or perpendicular) at the LC interface influences the LC structure throughout the droplet.24 As the simplest example for CLC droplets, the director field with tangentially aligning conditions49−51 exhibits the FrankPryce spherulitic texture with concentric rings. Another anchoring condition occurs when the mesogenic groups are perpendicular to the droplet surface. Unusual and unique structures causing conical flashlight and ring reflections have recently been reported for these droplets with perpendicular mesogen orientation at the LC interface.52 Recently, a microfluidic technique was used to overcome the sizepolydispersity bottleneck in the fabrication of LC droplets, 26408

DOI: 10.1021/acsami.6b09624 ACS Appl. Mater. Interfaces 2016, 8, 26407−26417

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ACS Applied Materials & Interfaces

Figure 1. Bright-field transmittance mode images of (a) CLCPVA and (b) CLCSDS droplets with ϕ = (i) 0.8, (ii) 1, (iii) 2, (iv) 5, and (v) 10 wt % chiral dopant.

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droplets were slowly injected into the middle inlet, and a continuous aqueous phase containing PVA (1 wt %), SDS (1 wt %), or PAA-bLCP (0.2 wt %) was injected into the other inlets in a direction perpendicular to that of the dispersed phase. The perpendicular phase streams met at a junction, and droplet formation took place when each stream crossed the neck of its channel. The resulting CLC droplets were extracted from the microchip and collected in a 25 × 25 × 3.5 mm3 storage reservoir, which was made by gluing a thin silicon rubber sheet onto the glass slide. GOx (or ChO)-Immobilized LC Droplets. The CLCPAA droplets, collected in a vial, were activated for 1 h with EDC·HCl: NHS (0.4 M: 0.1 M) in PBS buffer (pH 7.0). To obtain the GOx-immobilized CLC droplets (CLCPAA‑GOx droplets), we prepared a mixture of GOx (19 μM) and the activated CLCPAA droplets. The immobilization reaction occurred in this mixture for 12 h at 20 °C. Immobilization of GOx to PAA was confirmed using GOx labeled with rhodamine 6G (GOx−rhd), which was prepared as follows. To activate the carboxylic groups (−COOH) of GOx, this enzyme was dissolved (15.6 μM) in PBS buffer (pH 7.0) with 0.4 M EDC·HCl and 0.1 M NHS. This activation proceeded for 1 h. Next, rhodamine 6G (1 mg) was added to the solution, and the mixture was stirred for 12 h at 20 °C. We subsequently immobilized the GOx-rhd to the CLCPAA droplets using the same method that we had used to immobilize GOx to the CLCPAA droplets. After immobilization, the reacted solution was diluted with distilled water to remove the unreacted GOx-rhd, and the resulting CLCPAA‑GOx‑rhd droplets were examined using a fluorescent microscope. To prepare CLCPAA‑ChO, we repeated the above steps to immobilize ChO on the CLCPAA droplets. Measurements. The formation of CLC droplets on a chip was imaged using a high-speed digital video-recording camera (Motion BLIZT Cube4, Mikroton, Germany) with an inverted microscope (JSP-20T, Samwon, South Korea). GOx-rhd was examined using a UV−vis spectrometer (UV-2401PC, Shimadzu, Japan), and the presence of GOx-rhd on the CLCPAA‑GOx‑rhd droplets was confirmed by fluorescence microscopy (E600POL, Nikon Eclipse, Japan). We acquired 1H nuclear magnetic resonance (NMR) spectra of PAA-bLCP at 400 MHz (400 MHz, Bruker, Germany). The molecular weight was measured by gel permeation chromatography (GPC, Acme9000, Young Lin Instrument Co., Ltd.).

EXPERIMENTAL SECTION

Materials. MLC-2132 (Merck, U.K., nematic to isotropic transition temperature: 114.0 °C), (S)-4-cyano-4′-(2-methylbutyl)biphenyl (CB15) (Synthon, Germany), poly(vinyl alcohol) (PVA) (Yakuri, Japan), sodium dodecyl sulfate (SDS) (DC Chemical Co., Ltd. Korea), polysorbate 80 (Sigma−Aldrich), rhodamine 6G (Sigma−Aldrich), Nhydroxysuccinimide (NHS) (Sigma−Aldrich), N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide (EDC•HCl) (Sigma−Aldrich), pH buffer solutions (Samchun, Korea), glucose (Sigma−Aldrich), glucose oxidase (GOx, Sigma−Aldrich), cholesterol (Sigma−Aldrich), cholesterol oxidase (ChO, Sigma−Aldrich), galactose (Sigma−Aldrich), hemoglobin (Sigma−Aldrich), L-ascorbic acid (Sigma-Aldrich), and human serum (Sigma−Aldrich) were used as received. Using a previously reported method,30 we polymerized PAA-b-LCP. The Supporting Information (Scheme S1) contains details on the synthesis of PAA-b-LCP. Preparation of CLC droplets using microfluidics. Uniform CLC droplets were produced using PDMS-based microfluidic flowfocusing devices with MLC-2132 doped CB15. For the fabrication of the microfluidic flow-focusing devices, PDMS was prepared by mixing the prepolymer and cross-linker thoroughly at the recommended ratio of 10:1 (w/w). This mixture was degassed for 40 min in a desiccator to remove the remaining air bubbles. The final mixture was poured onto a structured silicon wafer mold, cured inside an oven at 65 °C for 4 h, and removed from the mold. This patterned piece of PDMS was bonded to a precleaned glass microscope slide using a short oxygen plasma treatment (46 s duration, Femto Science Inc., Korea). Figure S1 shows a schematic diagram of the microchip, with the dimensions of the microfluidics channel. The width of the inlet channels, the orifice width and length, and the width and height of the outlet channel were 110, 40, 40, 160, and 40 μm, respectively, and the depth of the channels was 100 μm throughout. The channel walls and chip assembly were made hydrophilic by treatment with APTES (2 wt % in ethanol) at 20 °C for 10 min, and a second treatment at 60 °C for 16 min. The microfluidic chip was mounted under an inverted biological microscope (Samwon NSI-100, South Korea). The liquid samples were supplied to the microfluidic device through flexible plastic tubings (Norton, USA, I.D. 0.51 mm, O.D. 1.52 mm) attached to the Fluiwell (Fluigent, France) containing the liquids. Flow rates were controlled using a pneumatic microfluidic flow rate control system (MFCS-EZ, Flow-Rate Platform and Flow-Rate Control Module, Fluigent) capable of pumping three fluids at a specified velocity. By pumping nitrogen gas at a finely controlled rate into the Fluiwell, the MFCS-EZ unit was used to pressurize the Fluiwell such that fluids began to flow through the tubes and into the device. The formation of on-chip droplets was imaged using an STC-TC83USB-AS camera (SenTech, Japan) attached to the inverted microscope. The droplets were observed under a polarized optical microscope (POM) (ANA006, Leitz, Germany) with crossed polarizers and a CCD camera (STC-TC83USB, Samwon, South Korea). The dispersed CLC

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RESULTS AND DISCUSSION

Preparation of CLC Droplets at Low Chiral Dopant Concentrations. When the content of chiral dopant in a CLC droplet (ϕ) is low, the droplet helical structure can be visualized through an optical microscope. PVA decoration on the CLC droplet causes tangential anchoring of CLC against the droplet surface. Figure 1a shows bright-field images of a CLCPVA droplet with ϕ = 0.8, 1, 2, 5, and 10 wt % chiral dopant. Helical structures with concentric rings are present, and 26409

DOI: 10.1021/acsami.6b09624 ACS Appl. Mater. Interfaces 2016, 8, 26407−26417

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ACS Applied Materials & Interfaces

Figure 2. (a, b) Bright-field reflection-mode and (c, d) POM (under crossed polarizers) images of (a, c) CLCPVA and (b, d) CLCSDS droplets at ϕ = (i) 30, (ii) 35, (iii) 40, (iv) 45, and (v) 50 wt %.

their pitches decrease as ϕ increases. When the helical twist axis in the CLC droplet has a radial orientation, the tangential orientation of the droplet surface matches the mesogen orientation, which is perpendicular to the helical twist axis. Thus, the PVA coating on the surface of the CLC droplet leads to concentric rings. The pitch (p) of the helix in the CLCPVA droplet is twice the distance between consecutive rings, due to n = −n, where n is the LC director. Figure S2 plots p as a function of 1/ϕ, with good linearity (r2 = 0.9914). The slope of this line is the inverse of the helical twisting power (HTP). Helical twisting power is a measure of the ability of the chiral dopant to twist the NLC defined by p = 1/(HTP[c]). The HTP is 7.08 (μm−1), which is similar to the previously reported value.59 An ionic surfactant, SDS, was used for homeotropic anchoring on the surfaces of the CLC droplets. A completely different CLC droplet structure is observed (Figure 1b) for droplets produced with 1 wt % SDS aqueous solution (CLCSDS droplets). The twist axis of the mesogenic groups is tangential in the homeotropic anchoring so that the direction of the twisting axis does not match the radial direction of the droplet. Thus, under this homeotropic anchoring condition on the CLC droplet surface, a symmetric concentric ring structure cannot be obtained. The CLCSDS droplet shows the surface and bulk orderings that cause the spiral lines and concentric rings in the image, respectively. The typical surface spiraling disclination, which was numerically predicted for short-pitch cholesterics in the case of droplets with perpendicular surface anchoring conditions, was observed in the CLCSDS droplets.60 The most striking difference between the bulk orientations of CLCPVA and CLCSDS droplets is the position of the center of the concentric rings. At all studied ϕs, this center shifts from the

droplet center for the CLCPVA droplet to off-center for the CLCSDS droplet. The p of CLCSDS is almost the same as that of CLCPVA. When ϕ is low (0.8 and 1 wt %, Figure 1b, i and (ii), the parallel layers connect to the curvature of the droplet surface and become similar to a series of nested cups. At ϕ = 2, 5, and 10 wt %, the cups become closed, giving rise to radial singularities. Figure S3 shows the bright-field transmittancemode, transmission-mode with cross polarizers, and bright-field reflection-mode images of CLCPVA droplets at ϕ = 12, 14, 16, and 18 wt %. The CLC droplets at this range do not show both the helical structure (due to its short pitch) and the reflection color (because the reflected light is out of visible range). PVA and SDS are known to cause parallel and perpendicular anchoring on the CLC surface, respectively. Therefore, the PVA- and SDS-coated CLC droplets were tested to find the characteristic CLC configuration by parallel and perpendicular anchoring on the CLC surface, respectively.13,14 Thus, the profound difference between the optical images of the CLCPVA droplets and those of the CLCSDS droplets could be used in sensor applications, if environmental changes could induce the LC orientation to change between parallel and perpendicular. Preparation of Cholesteric LC Droplets at High Chiral Dopant Concentrations. Figure 2a shows reflection mode bright-field images of CLCPVA droplets with ϕ = 30, 35, 40, 45, and 50 wt % chiral dopant, which were prepared using the same method as the CLCPVA droplets with low ϕs. The helical structure could not be observed because of the short pitch. However, the reflection mode bright field images show different colors at the center of the CLCPVA droplets, depending on the ϕ. These colors result from the periodic nature of the droplet superstructure. The wavelength of the color is directly 26410

DOI: 10.1021/acsami.6b09624 ACS Appl. Mater. Interfaces 2016, 8, 26407−26417

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Figure 3. Structural transition of CLCPVA droplets to CLCSDS droplets (i) 0, (ii) 4, (iii) 10, (iv) 18, and (v) 30 s after the addition of an aqueous SDS solution (5 wt %) to mixtures containing CLCPVA droplets at (a) ϕ = 2 and (b) ϕ = 45 wt % chiral dopant.

intermediate anchoring conditions (between 0° and 90°, parallel and perpendicular, respectively) at the LC-aqueous interface. Figure 2d shows the POM images of the CLCSDS prepared at different concentrations of the chiral dopant. A homeotropic orientation was observed, corresponding to the perpendicular alignment of the mesogen along the substrate. This perpendicular orientation of the mesogen molecule did not match with the tangent of the microsphere, which can lead towards the irregular patterns observed on the surface of the droplet. In particular, the presence of several randomly positioned flashlight spots in Figure 2b suggests that the several conical domains causing flashlight reflections were generated by the perpendicular anchoring conditions, as shown in Figure S4. Structural Transition of CLC Droplets from Parallel to Perpendicular Anchoring. We investigated the transient structures occurring during transitions from planar (tangential) to homeotropic (perpendicular) orientations in the CLC droplets. To observe the structural transition of a CLC droplet during its change from planar to homeotropic orientation, excess SDS solution (5 wt %) was added to the CLCPVA droplet reservoir. Movie SI 1 and Figure 3 show the structural transition of CLC droplets stabilized with PVA (1 wt %) at ϕ = 2 wt % chiral dopant (low concentration, Figure 3a) and 45 wt % chiral dopant (high concentration, Figure 3b). The CLCPVA droplet at ϕ = 2 wt % chiral dopant exhibited a clear helical structure before the SDS solution was added. This helical structure completely changed into a featureless droplet upon SDS addition. However, 10 s later, a texture resembling a series of nested cups emerged. This texture indicated that the CLC molecules were in an intermediate anchoring condition. The long axes of the CLC molecules were roughly perpendicular to the surface at the interfacial region, consistent with the result shown in Figure 1b. The CLCPVA droplets at ϕ = 45 wt % chiral dopant (high concentration) exhibited clear circular blue spots at their centers prior to the addition of SDS solution. These spots disappeared in 8 s after the SDS solution was added. New small, weak spots with a chicken skin-like appearance emerged (Figure 3b, (ii). These small spots may be attributable to the small conical domains. The number of spots decreased and their size increased with time, indicating that these domains were merging and growing. Thus, the chicken-skin and

proportional to the pitch (the length scale of one complete rotation of the director). For CLC mixtures, the spectral position of the reflection band gap is described as λb = np, meaning that the spectral position is governed by the average refractive index of the mixture (n) and the pitch length (p = 1/ ([c] × HTP)). The reflection wavelength could be varied by adjusting the chiral dopant concentration ([c]) through the equation λb = n/(HTP[c]). The observed colors of the CLCPVA droplets at ϕ = 30, 35, 40, 45, and 50 wt % chiral dopant were red, orange, yellow, bright blue, and blue, respectively. The λbs calculated from HTP and n (1.64 for MLC-2132) at ϕ = 30, 35, 40, 45, and 50 wt % were 779.8, 668.4, 584.8, 519.9, and 467.9 nm, respectively. These wavelengths are well matched with those of human-observed colors. At ϕ = 25 and 55 wt % chiral dopant, the central spot was not observed, because the wavelength was not in the range of the visible spectrum. In addition to the central spot, some additional radial lines and spots on the line connecting the two central spots were observed. The intensities and sizes of the additional spots are much weaker and smaller than those of the central spot. These additional spots and lines are attributed to lateral photonic cross-talk between the droplets.61,62When the incident light strike at an angle of 45° is reflected parallel to the substrate, and is then reflected again by the neighboring micro droplets at an angle of 45° perpendicular to the substrate. This reflection at different angles results in photonic cross-communication. Figure 2c shows the POM images of the CLCPVA prepared at different concentrations of chiral dopant. The images thus obtained show a cross pattern and a series of concentric rings around a small cross pattern at the center. These cross patterns and concentric rings are typical evidence of a radially oriented helical axis and suggest a macroscopic isotropic helical geometry of the helical structures. Figure 2b shows the reflection mode bright-field images of the CLCSDS droplets at ϕ = 30, 35, 40, 45, and 50 wt % chiral dopant, which were prepared using the same method as that used to prepare droplets with low ϕ. The central spots observed on the CLCPVA droplets disappeared, and strong flashlight-like spots were observed at the periphery of the CLCSDS droplet. This result is similar to the reported localized conical reflection (flashlight type)52 of the emulsion in water with surfactant, which led to the formation of spherulite-like CLC droplets induced by 26411

DOI: 10.1021/acsami.6b09624 ACS Appl. Mater. Interfaces 2016, 8, 26407−26417

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ACS Applied Materials & Interfaces

Figure 4. Bright-field images of CLCPAA droplets with (a) ϕ = 2 and (b) ϕ = 35 wt % chiral dopant, at pH 5 (i), 6 (ii), 7 (iii), 8 (iv), and 9 (v).

Scheme 1. Schematic Models of the CLC Configurations at (a) Low and (b, c) High pH

Figure 5. Bright-field reflection-mode images of CLCPAA droplets with ϕ = (a) 30, (b) 35, and (c) 40 wt %, at (i) 25, (ii) 30, (iii) 35, (iv) 40, and (v) 45 °C.

flashlight structures may be attributed to the weak and strong perpendicular anchoring conditions, respectively. More details about chicken-skin and flashlight structures are provided in Figure S5.

pH-Sensitive CLC Droplets Coated with PAA-b-LCP. To apply CLC droplets to biosensing, their surfaces should be coated with a material that responds to the external stimuli of interest, and this response should be able to change the CLC 26412

DOI: 10.1021/acsami.6b09624 ACS Appl. Mater. Interfaces 2016, 8, 26407−26417

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ACS Applied Materials & Interfaces

Figure 6. Bright-field images of CLCPAA‑GOx droplets (ϕ = 35 wt % chiral dopant) after injecting (a) glucose solutions at Cg = (i) 0.1, (ii) 0.2, (iii) 0.5, (iv) 1 μM; (b) (i) hemoglobin (100 μM), (ii) galactose (100 mM), (iii) L-ascorbic acid (100 μM), and (iv) cholesterol (100 μM) solutions; (c) (i) 500 × , (ii) 200 × , (iii) 100 × , and (iv) 10 × diluted human serum (original glucose concentration was approximately 40−140 mg/dL (0.022− 0.078 mM); (d) bright-field images of the CLCPAA‑ChO droplets (ϕ = 40 wt % chiral dopant) at pH 7 after adding the cholesterol solutions at Cc = (i) 0.625, (ii) 1.25, (iii) 2.5, and (iv) 25 μM.

droplet configuration. We implemented this design by decorating the surfaces of CLC droplets with the weak polyelectrolyte-containing block copolymer PAA-b-LCP. We named the resulting droplets as CLCPAA droplets. PAA is a weak polyelectrolyte. At low pH, PAA is protonated, has a neutral charge state, and as a bulk material, it shrinks. At high pH, PAA is deprotonated, has a negative charge state, and as a bulk material, it swells. The charged state of PAA favors a homeotropic orientation.30 Figure 4a shows the bright-field images of CLCPAA droplets at ϕ = 2 wt % chiral dopant under crossed polarizers at different pH. The closed, concentrically layered structure at low pH (pH 5, 6, and 7) changed to several open, layered, and divided domains at high pH (pH 8 and 9). When ϕ was high (ϕ = 35 wt % chiral dopant, Figure 4b), a clear yellow spot was observed at low pH (pH 5 and 6). At high pH (pH 7, 8, and 9), this central spot disappeared, and several small spots emerged at apparently random locations. Small red spots emerged from the small broken domain containing the remains of the helical structure. This broken domain may have been orientated with the layers parallel to the surface, causing the small reflection at the direction perpendicular to the layers as shown in Scheme 1. Thus, the helical structure of the

CLCPAA droplet is pH-sensitive. This pH-sensitive structure provides the basis for sensing pH-perturbing target analytes. Effect of Temperature. Figure 5 depicts the effect of temperature on the optical appearance of the CLCPAA droplets prepared at different concentrations of ϕ. The optical appearance of the CLC droplets was not affected by the change in the temperature from 25−45 °C. These results suggested that CLC droplets could be utilized for sensing purposes within a wide range of temperatures. Glucose and Cholesterol Biosensor. GOx and ChO can be immobilized via an EDC-NHS coupling reaction. To confirm successful immobilization, GOx was labeled with rhodamine 6G. Figure S6 shows the UV−vis absorption spectra of GOx, GOx-rhd, and rhodamine 6G. After labeling with rhodamine 6G, the spectrum of GOx-rhd exhibited a characteristic rhodamine 6G peak at 520 nm, indicating that the label had been successfully attached. GOx-rhd was subsequently immobilized on CLCPAA droplets via an additional EDC-NHS coupling reaction. The inset of Figure SI 6 shows the image obtained upon excitation of the rhodamine fluorophore at a wavelength of 365 nm. A clear green color was observed, indicating that immobilization of GOx occurred successfully on 26413

DOI: 10.1021/acsami.6b09624 ACS Appl. Mater. Interfaces 2016, 8, 26407−26417

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ACS Applied Materials & Interfaces Scheme 2. Cholesterol Oxidase Enzymatic Reaction

Figure 7. Bright-field images of the CLCPAA‑GOx droplet (ϕ = 35 wt % chiral dopant) at pH 7 (a) when injecting 100 μM glucose solution (i) 0, (ii) 2, (iii) 4, (iv) 6, (v) 8, (vi) 10, (vii) 12, and (viii) 14 d after the CLCPAA‑GOx droplets were freshly prepared, (b) (i) 0 (before), (ii) 4, (iii) 8, and (iv) 12 s after injecting 100 μM glucose solution, and the CLCPAA‑ChO droplet (ϕ = 40 wt % chiral dopant) at pH 7 (C) (i) 0 (before), (ii) 4, (iii) 8, and (iv) 12 s after injecting 100 μM cholesterol solution.

central spot at high Cgs (0.5 and 1 μM), indicating that the CLCPAA‑Gox droplet is suitable for use as a glucose biosensor, with a detection sensitivity of ∼0.5 μM.”This transition occurred because an enzymatic reaction produced gluconic acid as the glucose solution was introduced, which decreased the pH at the interface, which may be a reason leading to conversion to the planar configuration.14 One of other possible reason causing the configurational change of the CLC droplet might be the specific binding of glucose and cholesterol leading to the conformational disruption of immobilized PAA chains,

the surfaces of the CLCPAA droplets. The resulting CLC droplets were named as CLCPAA‑GOx droplets. 35 and 40 wt % of chiral dopant concentrations were utilized for the sensing of glucose and cholesterol, respectively. These concentrations of chiral dopant exhibited two distinct red and green colors. Therefore, this strategy could be utilized for multiple sensing systems. Figure 6a shows bright-field images of the CLCPAA‑GOx droplets (ϕ = 35 wt % chiral dopant) after adding glucose solutions at different concentrations (Cgs) at pH 7. The initial scattered red spots at low Cgs (0.1 and 0.2 μM) changed to a 26414

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(Figures 7a [i] to [vi]). However, the scattered spots of the CLCPAA‑GOx droplet did not change 10 d after the samples were freshly prepared (Figure 7a [vii] and [viii]), indicating that the CLCPAA‑GOx droplet is stable at least for 10 d. Next, the speed of glucose detection by the CLCPAA‑GOx droplet was tested. Figure 7b shows bright-field images of the CLCPAA‑GOx droplet at pH 7 at different time points after injecting 100 μM glucose solution. The scattered spots of the CLCPAA‑GOx droplet (Figure 7b [i]) changed to the central spot 4 s after 100 μM glucose solution was introduced (Figure 7b [ii]), indicating that the CLCPAA‑GOx droplet could detect glucose quite rapidly (within 4 s). Similarly, Figure 7c shows bright-field images of the CLCPAA‑ChO droplet at pH 7 at different times after injecting 100 μM cholesterol solution. The scattered spots of the CLCPAA‑ChO droplet (Figure 6c [i]) changed to the central spot 4 s after 100 μM cholesterol solution was introduced (Figure 7c [ii]), indicating that the CLCPAA‑ChO droplet could detect cholesterol with a very short response time (within 4 s). Thus, the speed of the CLCPAA‑GOx and CLCPAA‑ChO droplets is sufficient for biosensor applications. In order to test the reproducibility and accuracy of the sensor, the CLCPAA‑GOx and CLCPAA‑Cho droplets were alternatively exposed to glucose and cholesterol solution followed by subsequent washing with ∼10 mL of each PBS buffer (pH = 7) and DI water to reobtain the reference homeotropic state (data not shown). It was able to detect the glucose and cholesterol sample again. These results indicate that the CLCPAA‑GOx and CLCPAA‑Cho droplets can be used for multiple detections of glucose and cholesterol. Thus, the CLC droplets show high stability and activity, and capability of multiple usages through the covalent immobilization of the enzymes (GOx/ChO).

although the more detailed understanding of the mechanism is required. To evaluate the potential for interference from other biomaterials in human blood, hemoglobin (100 μM), galactose (100 μM), L-ascorbic acid (100 μM), and cholesterol (100 μM) aqueous solutions were added to the mixture (Figure 6b). The initial scattered red spots did not change after these biomaterials were added. In particular, the nonresponse for galactose, which has a similar structure as glucose (the main difference between them is the orientation of the hydroxyl group at the carbon), indicates that the CLCPAA‑GOx droplet responded specifically to glucose. Human serum, which contains 0.022−0.078 mM glucose, was also tested with water dilution, as shown in Figure 6c. The initial small, scattered red spots of the CLCPAA‑GOx droplet at low glucose concentrations (500 and 200 × dilutions) changed to a central spot at high glucose concentrations (100 and 10 × dilutions). The glucose solution at 100 and 10 × dilutions contains 0.5 and 5 μM of glucose, respectively, consistent with limit of glucose detection. Thus, the CLCPAA‑GOx droplet can be used to sense glucose in a real human blood sample, up to a limit of 100 × dilution with water. The CLCPAA‑ChO droplet was prepared similarly (see experimental section). Figure 6d shows brightfield images of CLCPAA‑ChO droplets with ϕ = 40 wt % chiral dopant. The dopant was chosen to distinguish between CLCPAA‑GOx and CLCPAA‑ChO droplets by their red or green color. We added cholesterol solutions of different concentrations (Ccs) at pH 7. The initial scattered green spots at low Ccs (0.625 and 1.25 μM) changed to a central spot at high Ccs (2.5 and 25 μM), indicating that the CLCPAA‑ChO droplet is suitable for use as a cholesterol biosensor, with a detection sensitivity of 2.5 μM. This transition occurs because an enzymatic reaction produces protons and cholest-4-ene-3-one as the cholesterol solution is introduced. The produced protons decrease the pH at the interface, leading to conversion to the planar configuration.63 Sekretaryova et al. studied the mechanism of electrochemical cholesterol sensors in detail and showed that the enzymatic reaction is vigorous after it starts. Wei et al. reported that the enzymatic reaction between cholesterol and cholesterol oxidase leads toward the formation of cholest-4-ene-3-one (D4-cholestenon) and is able to generate H+ as shown in Scheme 2, which could be detected by pH-sensitive LCs polymers. To confirm that the change in CLC configuration was due to the enzymatic reaction of GOx and ChO 100 μM of glucose and cholesterol solutions was injected into the cells without immobilizing GOx and ChO under otherwise similar conditions. A change in CLC droplets configuration was not observed, indicating that the configuration change was due to an enzymatic reactions. The optical response of the CLCPAA‑GOx/ChO was noted at low and high pH as shown in Figure S7. The planar and homeotropic orientations were observed at low (4−6) and high pH (7−9), respectively. These results are similar to the CLCPAA, indicating that the orientational transitions of CLCPAA‑GOx and CLCPAA‑ChO droplets are triggered by pH on the introduction of the specific analyte. Next, the stability of the CLCPAA‑GOx droplet was tested. Figure 7a shows bright-field images of the CLCPAA‑GOx droplet at pH 7 when injecting a 100 μM glucose solution various days after the CLCPAA‑GOx droplets were freshly prepared. The scattered spots of the CLCPAA‑GOx droplet changed to the central spot immediately after 100 μM glucose solution was introduced (Figure 7a [i]). This structural change happened 10 d after the CLCPAA‑GOx droplets were prepared

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CONCLUSION A central reflecting spot, many chicken-skin-like spots, and several flashlight-like spots of the CLC droplet with high ϕs represented planar (tangential), weak, and strong homeotropic anchoring conditions on the droplet surface, respectively. The pH-responsive and color-inducing CLCPAA droplets at high ϕs were used as a basic platform for developing new biosensors by immobilizing enzymes on the surface. The CLCPAA‑GOx and CLCPAA‑ChO droplets showed high sensitivity, good selectivity, and fast response to the presence of glucose and cholesterol. This detection method is novel because changes in the color pattern are observable with the naked eye, without crossed polarizers needed in NLC systems.

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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.6b09624. Additional materials as discussed in the text. (PDF)

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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 study was supported by the National Research Foundation of Korea (NRF-2011-0020264 and NRF2014R1A2A1A11050451). 26415

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