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A new strategy for imaging urease activity using liquid crystal droplet patterns formed on solid surfaces
Sensors and Actuators B 193 (2014) 770–773
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A new strategy for imaging urease activity using liquid crystal droplet patterns formed on solid surfaces Dingdong Liu, Chang-Hyun Jang ∗ Department of Chemistry, Gachon University, Seongnam-Si 461-701, Gyeonggi-Do, Republic of Korea
a r t i c l e
i n f o
Article history: Received 26 November 2013 Accepted 9 December 2013 Available online 18 December 2013 Keywords: Liquid crystals Droplet pattern Detection Enzymatic reaction Urease
a b s t r a c t In this study, we demonstrate a simple and label-free method for imaging urease activity using liquid crystal (LC) droplet patterns on solid surfaces. The LC droplet patterns were spontaneously formed by spreading stearic acid doped-LCs dissolved in organic solvents on glass microscope slides. The LC droplets displayed a bright appearance in urea or urease solution, while dark cross images were exhibited when LC droplets contacted an aqueous mixture of urease and urea, indicating an orientational transition of LC molecules from a planar to a perpendicular state. The enzymatic reaction between urea and urease produced ammonia that could be hydrolyzed into ammonium and hydroxide ions. Due to the increase of pH, the carboxylic acids were deprotonated and self-assembled at the aqueous/LC interface, which induced an orientational transition of LC molecules in the droplet. These results indicate that the surfaceanchored LC droplets with dopants show high promise for developing LC pattern-based sensing devices for label-free detection of biological events. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In recent years, liquid crystalline materials have attracted great interest for amplifying and transducing biomolecular events into optical signals that are visible by the naked eye under crossed polarizers [1–9]. The orientation of liquid crystals (LCs) responds rapidly to chemical and biological events with high spatial resolution and sensitivity [10]. Thus, LC-based sensors may be effective, simple, and promising tools for detection applications [11,12] that do not require labeled analytes [13], laborious techniques [14], or complex instrumentation [15]. It is well known that chemical and biological events can be amplified and transduced by nematic LCs at the fluid aqueous/LC interfaces [16,17]. For such sensing systems, nematic LCs are always deposited into the holes of a metallic grid supported on a glass slide, which can prevent the hydrophobic 5CB from dewetting [18,19]. However, the grids are usually not completely flat, which may induce a distorted liquid crystal interface [20]. In addition, preparation of the optical cells with chambers that are used in this sensing system is relatively time-consuming. We recently reported spontaneously formed micrometer-scale LC droplets, which have distinguishable optical textures, supported on solid surfaces [21]. The LC droplets were formed by simply
∗ Corresponding author at: Kyungwon University, College of Bionano Technology, San 65 Bokjeong-Dong, Sujeong-Gu Seongnam, Gyeonggi 461-701, Republic of Korea. Tel.: +82 31 750 8555. E-mail address: [email protected] (C.-H. Jang). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.12.033
spreading LCs dissolved in organic solvents onto the glass slide surfaces. This system does not require the use of a metallic grid and chamber. Furthermore, the surface-anchored LC droplet patterns have large surface areas that may be sensitive to the presence of analytes. Urease, which belongs to the superfamily of amidohydrolases and phosphotriestreases [22], is produced in large amounts by Helicobacter pylori (Hp) [23]. The detection of urease is often used as an efficient and effective method to assess the presence of Hp in clinical samples [24,25], which is important to human health and safety [26]. We previously reported an LC-based sensor for the detection of urease at aqueous/LC interfaces using UV-treated 5CB [27]. The optical image of the LCs changed from bright-to-dark due to the enzymatic hydrolysis of urea by urease. However, this was a relatively time-consuming and complex system. Accordingly, it is still necessary to develop new strategies for the determination of urease activity. In this study, we demonstrate a simple and fast method for imaging urease activity using liquid crystal (LC) droplet patterns on solid surfaces. The LC droplets displayed a bright appearance (planar orientation) (Fig. 1A) in urea or urease solution, while dark crosses (perpendicular orientation) (Fig. 1B) were exhibited when LC droplets were in contact with an aqueous mixture of urease and urea. The enzymatic reaction between urea and urease produced ammonia that could be hydrolyzed into ammonium and hydroxide ions. Due to the increase of pH, the carboxylic acids were deprotonated and self-assembled at the aqueous/LC interface, inducing an orientational transition of LC molecules in the droplet. These results indicate that the LC droplets have great potential for developing LC
D. Liu, C.-H. Jang / Sensors and Actuators B 193 (2014) 770–773
Fig. 1. Schematic illustration of the orientation of LCs before and after the addition of urease and urea mixture onto the stearic acid-decorated LC droplet surface: (A) planar orientation, (B) perpendicular orientation of 5CB at the aqueous/LC droplets interface.
pattern-based sensing devices for label-free detection of biological events.
771
Fig. 2. Polarized light microscopy images of the patterns of stearic acid-doped 5CB droplets that were: (A) exposed to the air, (B) immersed into DI water.
3. Results and discussion 3.1. Orientational behaviors of acid-doped LC droplet pattern induced by different pH solutions
2. Experimental 2.1. Materials Nematic liquid crystal (LC) 4-cyano-4 -pentylbi-phenyl (5CB) manufactured by BDH Chemicals was obtained from EM industries (Hawthorne, NY). Premium glass microscope slides were purchased from Fisher Scientific (Pittsburgh, PA). n-Heptane (anhydrous) was purchased from Daejung Chemicals & Metals Co., Ltd. (South Korea). Sulfuric acid, hydrogen peroxide (30% w/v), octyltrichlorosilane (OTS), urease, urea, sodium hydroxide, and phosphate buffered saline (PBS) (10 mM phosphate, 138 mM NaCl, 2.7 mM KCl; pH 7.4) were obtained from Sigma–Aldrich. All aqueous solutions were prepared with deionized water (18 M cm) using a Milli-Q water purification system (Millipore, Bedford, MA). 2.2. Treatment of glass microscope slides Glass microscope slides were cleaned as previously described [21,27] using piranha solution (70% H2 SO4 /30% H2 O2 ) for 30 min at 80 ◦ C (caution: piranha solution reacts violently with organic materials and should be handled with extreme caution; do not store the solution in closed containers). The slides were then rinsed with water, ethanol, and methanol, respectively, after which they were dried under a stream of gaseous N2 , followed by heating to 120 ◦ C overnight prior to OTS deposition. Next, the slides were immersed in OTS/n-heptane solution for 30 min, after which they were rinsed with methylene chloride and dried under a stream of N2 . 2.3. Preparation of large scale LC droplet patterns The micro-scale patterns of LC droplets supported on OTStreated glass slides were formed by dropping 1 l 1% (V/V) 5CB doped with stearic acid dissolved in heptane (anhydrous) onto the glass surface. After evaporation of the organic solvent, 1 l aqueous solutions of interest were introduced onto the LC droplet patterns at room temperature. 2.4. Examination of LC optical textures A polarized light microscope (ECLIPSE LV100POL, Nikon, Tokyo, Japan) was used to observe optical textures of 5CB with the polarized light transmitting through LC droplet patterns on the glass slides. All images were obtained using a 10× objective lens between crossed polarizers at room temperature. The images were then captured by a digital camera (DS- 2Mv, Nikon, Tokyo, Japan) attached to a polarized light microscope with a resolution of 1600 × 1200 pixels, a gain of 1.00 × and a shutter speed of 1/10 s.
We recently reported spontaneously formed liquid crystal droplet patterns with large surface areas on glass microscope slides. In addition, 5CB doped with functional molecules have shown high utility in development of a variety of LC-based sensors. Here, we investigated whether the stearic acid-doped LC droplet pattern could be used to detect pH-related chemical and biological events. First, LC droplet patterns were formed by spreading stearic acid doped-LCs dissolved in heptane on glass microscope slides. A dark cross image (Fig. 2A) was demonstrated upon polarized microscopy after evaporation of heptane due to the homeotropic alignment of LCs at the air/LC interface and LC/OTS glass surface interface. These findings were consistent with those of a previous study in which pure 5CB droplets on OTS-treated glass adopted a dark cross appearance [21]. We then examined the optical response of LCs after introducing DI water onto the droplet pattern and observed a bright appearance (Fig. 2B), which was attributed to the planar orientation of LC molecules at the aqueous/LC droplet interface. In a previous study, 4 -pentyl-biphenyl-4-carboxylic acid (PBA)doped 5CB was used to screen changes in pH in sodium phosphate buffer (PBS) at the aqueous/LC interface. When pH increased from 6.0 to 7.0, a bright-to-dark shift in the optical response was observed. PBA-doped 5CB was used to screen pH variations in sodium phosphate buffer (PBS) at the aqueous/LC interface [7]. A bright-to-dark change in the optical appearance indicating a planar-to-homeotropic transition of LCs was observed when pH was increased. These findings indicated that the spontaneously formed acid-doped LC droplets pattern on the glass surface had great potential for use in detecting pH-related reactions due to the large surface area and simple preparation procedures. We predicted that distinct optical responses of LCs might be obtained when the stearic acid doped-LC droplet patterns were immersed into different pH solutions. A series of PBS solutions with different pH were transferred onto the LC droplet patterns. When 1 l of PBS solution with a pH of 6 was introduced onto the acid-doped LC droplets pattern, a bright appearance (Fig. 3A) in optical response was exhibited, indicating a planar orientation of the LCs at the aqueous/LC droplet interface. This result was the same as the optical response of LC droplets immersed into DI water. In contrast, the LCs adopted dark cross appearance (Fig. 3B and C) when PBS solutions with a pH of 8 and 10 were dispensed onto the acid doped LC droplets, corresponding to a homeotropic alignment of LCs at the aqueous/LC droplet interface. Only bright images were obtained (data not shown) when the above experiments were conducted using the pure 5CB droplets pattern. Based on the above results, we also confirmed that the pH of aqueous solutions could influence the orientational behaviors of LC molecules at the aqueous/LC droplets interface. The pH of the buffer affected the deprotonation of the
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Fig. 3. Polarized light microscopy images of the patterns of stearic acid-doped 5CB droplets that were in contact with different pH PBS solutions: (A) pH 6, (B) pH 8, and (C) pH 10.
Fig. 4. Polarized light microscopy images of the patterns of stearic acid doped 5CB droplets that were in contact with: (A) a mixture of urease and urea solution, (B) 0.5 M urea solution, and (C) 0.1 mg/ml urease solution.
carboxylic acid molecules. These findings indicate that there may not be sufficient carboxylic acid deprotonated at the aqueous/LC droplet interface when PBS with a pH of 6 is introduced. Therefore, the LCs still adopted a planar orientation, which resulted in a bright appearance in the optical response. However, in the case the PBS solutions with pH values of 8 and 10, increases in pH led to the perpendicular orientation of the LC; thus, a dark cross image was obtained. 3.2. Imaging enzymatic interactions between urease and urea Previous studies have indicated that the enzymatic reaction between urea and urease produces ammonia that can be hydrolyzed into ammonium and hydroxide ions, which could lead to an increase in pH. In our system, we predicted that it might be feasible to determine the enzymatic events between urease and urea using the acid-doped LC droplet patterns formed on the OTStreated glass. We first tested an aqueous mixture of urease and urea that had been pre-incubated for 30 min at room temperature. A dark cross appearance (Fig. 4A) was observed immediately when the aqueous mixture was transferred onto the stearic acid-doped LC droplet pattern, indicating a homeotropic state of LCs at the aqueous/LC droplet interface. Due to the enzymatic reaction between urease and urea, ammonia, which can be hydrolyzed into ammonium and hydroxide ions, was produced. Owing to the increase of pH in the
aqueous solution, more carboxylic acid molecules protonated and self-assembled at the aqueous/LC droplet interface, resulting in a dark cross image coupled to the homeotropic state of LCs. The optical responses of the LC droplet patterns in the control experiments with only urea or urease solution were also examined to confirm that the dark cross image of the surface-anchored LC droplet was due to the enzymatic reaction between urease and urea in the aqueous phase. Bright appearances (Fig. 4B and C) suggesting a planar state of the LCs at the aqueous/LC droplet interface were obtained in both cases. The pH of the aqueous solutions remained the same in the absence of the enzymatic reactions between urease and urea. Thus, the deprotonated carboxylic acid molecules could not form a stable monolayer. Correspondingly, LCs adopted a planar orientation at the aqueous/LC droplet interface, exhibiting a bright appearance in the optical response. These results indicate that the acid-doped LC pattern could be used to monitor the enzymatic events between urease and urea. In addition, when compared with other techniques for measuring urease activity, the LC droplet pattern system for the determination of urease activity is simple, fast, and label-free. 3.3. Detection limit for the enzymatic activity of urease After confirming the feasibility of determining the enzymatic event between urease and urea using the LC droplet patterns, we examined the detection limit of urease. To determine the sensitivity
Fig. 5. Polarized light microscopy images of the patterns of stearic acid doped 5CB droplets that were in contact with a mixture of (A) 0.1 mg/ml urease and 0.5 M urea pre-incubated for 3 min, (B) 10 g/ml urease and 0.5 M urea pre-incubated for 30 min, and (C) 1 g/ml urease and 0.5 M urea pre-incubated for 90 min.
D. Liu, C.-H. Jang / Sensors and Actuators B 193 (2014) 770–773
of our method, urea with a concentration of 0.5 M was mixed with urease at different concentrations and pre-incubated for different time periods before the aqueous mixtures were transferred onto the acid doped-LC droplet pattern. We then incubated an aqueous mixture of 0.5 M urea and 0.1 mg/ml urease for 3 min at room temperature, which produced a completely dark cross image (Fig. 5A) immediately, indicating that the LCs adopted a homeotropic state at the aqueous/LC droplet interface. When an aqueous mixture of urea and 10 g/ml urease that had been pre-incubated for 30 min was introduced onto the acid-doped LC droplet pattern, a partly bright appearance was observed; however, the image gradually became dark. A nearly dark cross appearance (Fig. 5B) was obtained within 5 min, suggesting a planar-to-perpendicular anchoring transition of the LCs. These results indicate that an aqueous solution of urease with a concentration of 10 g/ml was sufficient to induce a change in the orientation of LCs. Next, a mixture of urea and 1 g/ml urease that had been pre-incubated for 90 min was contacted with the acid-doped LC pattern. When this was conducted, a bright appearance (Fig. 5C) was demonstrated. However, the textures of the LC droplet pattern were distinct from those obtained in DI water, urease solution or urea solution. As shown in Fig. 5C, a subtle brightto-dark transition in the optical texture was obtained, which might represent the orientational transition of a small amount of 5CB molecules at the aqueous/LC droplet interface. As described in a previous study involving LC droplet patterns, these findings might be due to the gradual evaporation of water from the aqueous phase that was in contact with the surface-anchored LC droplets. The increased concentration of surfactant in the aqueous phase could help improve the sensitivity of the sensing device. Taken together, these findings suggest that the detection limit for urease using this sensing system was about 1 g/ml. 4. Conclusion In summary, we demonstrated a simple label-free method for imaging urease activity using liquid crystal (LC) droplet patterns on solid surfaces. The LC droplets displayed a bright appearance in urea or urease solution; while dark crosses were exhibited when LC droplets were contacted with an aqueous mixture of urease and urea. The enzymatic reaction between urea and urease induced an orientational transition of LC molecules from a planar to a perpendicular state at the LC droplet interface, which produced distinct optical textures. These results indicate that the surface-anchored LC droplets pattern show high promise for simple and rapid label-free detection of biochemical events. Acknowledgement This work was supported by the Gachon University Research Fund of 2013 (GCU-2013-R387). References [1] V.K. Gupta, J.J. Skaife, T.B. Dubrovsky, N.L. Abbott, Optical amplification of ligand-receptor binding using liquid crystals, Science 279 (1998) 2077–2080. [2] J.S. Park, S. Teren, W.H. Tepp, D.J. Beebe, E.A. Johnson, N.L. Abbott, Formation of oligopeptide-based polymeric membranes at interfaces between aqueous phases and thermotropic liquid crystals, Chem. Mater. 18 (2006) 6147–6151. [3] A.D. Price, D.K. Schwartz, DNA hybridization-induced reorientation of liquid crystal anchoring at the nematic liquid crystal/aqueous interface, J. Am. Chem. Soc. 130 (2008) 8188–8194. [4] A. Hussain, A.S. Pina, A.C.A. Roque, Bio-recognition and detection using liquid crystals, Biosens. Bioelectron. 25 (2009) 1–8.
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Biographies Dingdong Liu received her M.S. degree in Chemistry in 2013 from Gachon University, Seongnam, South Korea. Her areas of research interest are anchoring and phase transition of liquid crystals to biomolecular recognition events occurring at the aqueous-LC interface. Chang-Hyun Jang is a professor in Chemistry Department, Gachon University, Seongnam, South Korea. His academic interests are liquid crystals-based biosensors, biological interactions at nanostructured interfaces, nanofabrication of proteins and particles on self-assembled monolayers, surfactant technology, and microencapsulation.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A new strategy for imaging urease activity using liquid crystal droplet patterns formed on solid surfaces Dingdong Liu, Chang-Hyun Jang ∗ Department of Chemistry, Gachon University, Seongnam-Si 461-701, Gyeonggi-Do, Republic of Korea
a r t i c l e
i n f o
Article history: Received 26 November 2013 Accepted 9 December 2013 Available online 18 December 2013 Keywords: Liquid crystals Droplet pattern Detection Enzymatic reaction Urease
a b s t r a c t In this study, we demonstrate a simple and label-free method for imaging urease activity using liquid crystal (LC) droplet patterns on solid surfaces. The LC droplet patterns were spontaneously formed by spreading stearic acid doped-LCs dissolved in organic solvents on glass microscope slides. The LC droplets displayed a bright appearance in urea or urease solution, while dark cross images were exhibited when LC droplets contacted an aqueous mixture of urease and urea, indicating an orientational transition of LC molecules from a planar to a perpendicular state. The enzymatic reaction between urea and urease produced ammonia that could be hydrolyzed into ammonium and hydroxide ions. Due to the increase of pH, the carboxylic acids were deprotonated and self-assembled at the aqueous/LC interface, which induced an orientational transition of LC molecules in the droplet. These results indicate that the surfaceanchored LC droplets with dopants show high promise for developing LC pattern-based sensing devices for label-free detection of biological events. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In recent years, liquid crystalline materials have attracted great interest for amplifying and transducing biomolecular events into optical signals that are visible by the naked eye under crossed polarizers [1–9]. The orientation of liquid crystals (LCs) responds rapidly to chemical and biological events with high spatial resolution and sensitivity [10]. Thus, LC-based sensors may be effective, simple, and promising tools for detection applications [11,12] that do not require labeled analytes [13], laborious techniques [14], or complex instrumentation [15]. It is well known that chemical and biological events can be amplified and transduced by nematic LCs at the fluid aqueous/LC interfaces [16,17]. For such sensing systems, nematic LCs are always deposited into the holes of a metallic grid supported on a glass slide, which can prevent the hydrophobic 5CB from dewetting [18,19]. However, the grids are usually not completely flat, which may induce a distorted liquid crystal interface [20]. In addition, preparation of the optical cells with chambers that are used in this sensing system is relatively time-consuming. We recently reported spontaneously formed micrometer-scale LC droplets, which have distinguishable optical textures, supported on solid surfaces [21]. The LC droplets were formed by simply
∗ Corresponding author at: Kyungwon University, College of Bionano Technology, San 65 Bokjeong-Dong, Sujeong-Gu Seongnam, Gyeonggi 461-701, Republic of Korea. Tel.: +82 31 750 8555. E-mail address: [email protected] (C.-H. Jang). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.12.033
spreading LCs dissolved in organic solvents onto the glass slide surfaces. This system does not require the use of a metallic grid and chamber. Furthermore, the surface-anchored LC droplet patterns have large surface areas that may be sensitive to the presence of analytes. Urease, which belongs to the superfamily of amidohydrolases and phosphotriestreases [22], is produced in large amounts by Helicobacter pylori (Hp) [23]. The detection of urease is often used as an efficient and effective method to assess the presence of Hp in clinical samples [24,25], which is important to human health and safety [26]. We previously reported an LC-based sensor for the detection of urease at aqueous/LC interfaces using UV-treated 5CB [27]. The optical image of the LCs changed from bright-to-dark due to the enzymatic hydrolysis of urea by urease. However, this was a relatively time-consuming and complex system. Accordingly, it is still necessary to develop new strategies for the determination of urease activity. In this study, we demonstrate a simple and fast method for imaging urease activity using liquid crystal (LC) droplet patterns on solid surfaces. The LC droplets displayed a bright appearance (planar orientation) (Fig. 1A) in urea or urease solution, while dark crosses (perpendicular orientation) (Fig. 1B) were exhibited when LC droplets were in contact with an aqueous mixture of urease and urea. The enzymatic reaction between urea and urease produced ammonia that could be hydrolyzed into ammonium and hydroxide ions. Due to the increase of pH, the carboxylic acids were deprotonated and self-assembled at the aqueous/LC interface, inducing an orientational transition of LC molecules in the droplet. These results indicate that the LC droplets have great potential for developing LC
D. Liu, C.-H. Jang / Sensors and Actuators B 193 (2014) 770–773
Fig. 1. Schematic illustration of the orientation of LCs before and after the addition of urease and urea mixture onto the stearic acid-decorated LC droplet surface: (A) planar orientation, (B) perpendicular orientation of 5CB at the aqueous/LC droplets interface.
pattern-based sensing devices for label-free detection of biological events.
771
Fig. 2. Polarized light microscopy images of the patterns of stearic acid-doped 5CB droplets that were: (A) exposed to the air, (B) immersed into DI water.
3. Results and discussion 3.1. Orientational behaviors of acid-doped LC droplet pattern induced by different pH solutions
2. Experimental 2.1. Materials Nematic liquid crystal (LC) 4-cyano-4 -pentylbi-phenyl (5CB) manufactured by BDH Chemicals was obtained from EM industries (Hawthorne, NY). Premium glass microscope slides were purchased from Fisher Scientific (Pittsburgh, PA). n-Heptane (anhydrous) was purchased from Daejung Chemicals & Metals Co., Ltd. (South Korea). Sulfuric acid, hydrogen peroxide (30% w/v), octyltrichlorosilane (OTS), urease, urea, sodium hydroxide, and phosphate buffered saline (PBS) (10 mM phosphate, 138 mM NaCl, 2.7 mM KCl; pH 7.4) were obtained from Sigma–Aldrich. All aqueous solutions were prepared with deionized water (18 M cm) using a Milli-Q water purification system (Millipore, Bedford, MA). 2.2. Treatment of glass microscope slides Glass microscope slides were cleaned as previously described [21,27] using piranha solution (70% H2 SO4 /30% H2 O2 ) for 30 min at 80 ◦ C (caution: piranha solution reacts violently with organic materials and should be handled with extreme caution; do not store the solution in closed containers). The slides were then rinsed with water, ethanol, and methanol, respectively, after which they were dried under a stream of gaseous N2 , followed by heating to 120 ◦ C overnight prior to OTS deposition. Next, the slides were immersed in OTS/n-heptane solution for 30 min, after which they were rinsed with methylene chloride and dried under a stream of N2 . 2.3. Preparation of large scale LC droplet patterns The micro-scale patterns of LC droplets supported on OTStreated glass slides were formed by dropping 1 l 1% (V/V) 5CB doped with stearic acid dissolved in heptane (anhydrous) onto the glass surface. After evaporation of the organic solvent, 1 l aqueous solutions of interest were introduced onto the LC droplet patterns at room temperature. 2.4. Examination of LC optical textures A polarized light microscope (ECLIPSE LV100POL, Nikon, Tokyo, Japan) was used to observe optical textures of 5CB with the polarized light transmitting through LC droplet patterns on the glass slides. All images were obtained using a 10× objective lens between crossed polarizers at room temperature. The images were then captured by a digital camera (DS- 2Mv, Nikon, Tokyo, Japan) attached to a polarized light microscope with a resolution of 1600 × 1200 pixels, a gain of 1.00 × and a shutter speed of 1/10 s.
We recently reported spontaneously formed liquid crystal droplet patterns with large surface areas on glass microscope slides. In addition, 5CB doped with functional molecules have shown high utility in development of a variety of LC-based sensors. Here, we investigated whether the stearic acid-doped LC droplet pattern could be used to detect pH-related chemical and biological events. First, LC droplet patterns were formed by spreading stearic acid doped-LCs dissolved in heptane on glass microscope slides. A dark cross image (Fig. 2A) was demonstrated upon polarized microscopy after evaporation of heptane due to the homeotropic alignment of LCs at the air/LC interface and LC/OTS glass surface interface. These findings were consistent with those of a previous study in which pure 5CB droplets on OTS-treated glass adopted a dark cross appearance [21]. We then examined the optical response of LCs after introducing DI water onto the droplet pattern and observed a bright appearance (Fig. 2B), which was attributed to the planar orientation of LC molecules at the aqueous/LC droplet interface. In a previous study, 4 -pentyl-biphenyl-4-carboxylic acid (PBA)doped 5CB was used to screen changes in pH in sodium phosphate buffer (PBS) at the aqueous/LC interface. When pH increased from 6.0 to 7.0, a bright-to-dark shift in the optical response was observed. PBA-doped 5CB was used to screen pH variations in sodium phosphate buffer (PBS) at the aqueous/LC interface [7]. A bright-to-dark change in the optical appearance indicating a planar-to-homeotropic transition of LCs was observed when pH was increased. These findings indicated that the spontaneously formed acid-doped LC droplets pattern on the glass surface had great potential for use in detecting pH-related reactions due to the large surface area and simple preparation procedures. We predicted that distinct optical responses of LCs might be obtained when the stearic acid doped-LC droplet patterns were immersed into different pH solutions. A series of PBS solutions with different pH were transferred onto the LC droplet patterns. When 1 l of PBS solution with a pH of 6 was introduced onto the acid-doped LC droplets pattern, a bright appearance (Fig. 3A) in optical response was exhibited, indicating a planar orientation of the LCs at the aqueous/LC droplet interface. This result was the same as the optical response of LC droplets immersed into DI water. In contrast, the LCs adopted dark cross appearance (Fig. 3B and C) when PBS solutions with a pH of 8 and 10 were dispensed onto the acid doped LC droplets, corresponding to a homeotropic alignment of LCs at the aqueous/LC droplet interface. Only bright images were obtained (data not shown) when the above experiments were conducted using the pure 5CB droplets pattern. Based on the above results, we also confirmed that the pH of aqueous solutions could influence the orientational behaviors of LC molecules at the aqueous/LC droplets interface. The pH of the buffer affected the deprotonation of the
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Fig. 3. Polarized light microscopy images of the patterns of stearic acid-doped 5CB droplets that were in contact with different pH PBS solutions: (A) pH 6, (B) pH 8, and (C) pH 10.
Fig. 4. Polarized light microscopy images of the patterns of stearic acid doped 5CB droplets that were in contact with: (A) a mixture of urease and urea solution, (B) 0.5 M urea solution, and (C) 0.1 mg/ml urease solution.
carboxylic acid molecules. These findings indicate that there may not be sufficient carboxylic acid deprotonated at the aqueous/LC droplet interface when PBS with a pH of 6 is introduced. Therefore, the LCs still adopted a planar orientation, which resulted in a bright appearance in the optical response. However, in the case the PBS solutions with pH values of 8 and 10, increases in pH led to the perpendicular orientation of the LC; thus, a dark cross image was obtained. 3.2. Imaging enzymatic interactions between urease and urea Previous studies have indicated that the enzymatic reaction between urea and urease produces ammonia that can be hydrolyzed into ammonium and hydroxide ions, which could lead to an increase in pH. In our system, we predicted that it might be feasible to determine the enzymatic events between urease and urea using the acid-doped LC droplet patterns formed on the OTStreated glass. We first tested an aqueous mixture of urease and urea that had been pre-incubated for 30 min at room temperature. A dark cross appearance (Fig. 4A) was observed immediately when the aqueous mixture was transferred onto the stearic acid-doped LC droplet pattern, indicating a homeotropic state of LCs at the aqueous/LC droplet interface. Due to the enzymatic reaction between urease and urea, ammonia, which can be hydrolyzed into ammonium and hydroxide ions, was produced. Owing to the increase of pH in the
aqueous solution, more carboxylic acid molecules protonated and self-assembled at the aqueous/LC droplet interface, resulting in a dark cross image coupled to the homeotropic state of LCs. The optical responses of the LC droplet patterns in the control experiments with only urea or urease solution were also examined to confirm that the dark cross image of the surface-anchored LC droplet was due to the enzymatic reaction between urease and urea in the aqueous phase. Bright appearances (Fig. 4B and C) suggesting a planar state of the LCs at the aqueous/LC droplet interface were obtained in both cases. The pH of the aqueous solutions remained the same in the absence of the enzymatic reactions between urease and urea. Thus, the deprotonated carboxylic acid molecules could not form a stable monolayer. Correspondingly, LCs adopted a planar orientation at the aqueous/LC droplet interface, exhibiting a bright appearance in the optical response. These results indicate that the acid-doped LC pattern could be used to monitor the enzymatic events between urease and urea. In addition, when compared with other techniques for measuring urease activity, the LC droplet pattern system for the determination of urease activity is simple, fast, and label-free. 3.3. Detection limit for the enzymatic activity of urease After confirming the feasibility of determining the enzymatic event between urease and urea using the LC droplet patterns, we examined the detection limit of urease. To determine the sensitivity
Fig. 5. Polarized light microscopy images of the patterns of stearic acid doped 5CB droplets that were in contact with a mixture of (A) 0.1 mg/ml urease and 0.5 M urea pre-incubated for 3 min, (B) 10 g/ml urease and 0.5 M urea pre-incubated for 30 min, and (C) 1 g/ml urease and 0.5 M urea pre-incubated for 90 min.
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of our method, urea with a concentration of 0.5 M was mixed with urease at different concentrations and pre-incubated for different time periods before the aqueous mixtures were transferred onto the acid doped-LC droplet pattern. We then incubated an aqueous mixture of 0.5 M urea and 0.1 mg/ml urease for 3 min at room temperature, which produced a completely dark cross image (Fig. 5A) immediately, indicating that the LCs adopted a homeotropic state at the aqueous/LC droplet interface. When an aqueous mixture of urea and 10 g/ml urease that had been pre-incubated for 30 min was introduced onto the acid-doped LC droplet pattern, a partly bright appearance was observed; however, the image gradually became dark. A nearly dark cross appearance (Fig. 5B) was obtained within 5 min, suggesting a planar-to-perpendicular anchoring transition of the LCs. These results indicate that an aqueous solution of urease with a concentration of 10 g/ml was sufficient to induce a change in the orientation of LCs. Next, a mixture of urea and 1 g/ml urease that had been pre-incubated for 90 min was contacted with the acid-doped LC pattern. When this was conducted, a bright appearance (Fig. 5C) was demonstrated. However, the textures of the LC droplet pattern were distinct from those obtained in DI water, urease solution or urea solution. As shown in Fig. 5C, a subtle brightto-dark transition in the optical texture was obtained, which might represent the orientational transition of a small amount of 5CB molecules at the aqueous/LC droplet interface. As described in a previous study involving LC droplet patterns, these findings might be due to the gradual evaporation of water from the aqueous phase that was in contact with the surface-anchored LC droplets. The increased concentration of surfactant in the aqueous phase could help improve the sensitivity of the sensing device. Taken together, these findings suggest that the detection limit for urease using this sensing system was about 1 g/ml. 4. Conclusion In summary, we demonstrated a simple label-free method for imaging urease activity using liquid crystal (LC) droplet patterns on solid surfaces. The LC droplets displayed a bright appearance in urea or urease solution; while dark crosses were exhibited when LC droplets were contacted with an aqueous mixture of urease and urea. The enzymatic reaction between urea and urease induced an orientational transition of LC molecules from a planar to a perpendicular state at the LC droplet interface, which produced distinct optical textures. These results indicate that the surface-anchored LC droplets pattern show high promise for simple and rapid label-free detection of biochemical events. Acknowledgement This work was supported by the Gachon University Research Fund of 2013 (GCU-2013-R387). References [1] V.K. Gupta, J.J. Skaife, T.B. Dubrovsky, N.L. Abbott, Optical amplification of ligand-receptor binding using liquid crystals, Science 279 (1998) 2077–2080. [2] J.S. Park, S. Teren, W.H. Tepp, D.J. Beebe, E.A. Johnson, N.L. Abbott, Formation of oligopeptide-based polymeric membranes at interfaces between aqueous phases and thermotropic liquid crystals, Chem. Mater. 18 (2006) 6147–6151. [3] A.D. Price, D.K. Schwartz, DNA hybridization-induced reorientation of liquid crystal anchoring at the nematic liquid crystal/aqueous interface, J. Am. Chem. Soc. 130 (2008) 8188–8194. [4] A. Hussain, A.S. Pina, A.C.A. Roque, Bio-recognition and detection using liquid crystals, Biosens. Bioelectron. 25 (2009) 1–8.
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Biographies Dingdong Liu received her M.S. degree in Chemistry in 2013 from Gachon University, Seongnam, South Korea. Her areas of research interest are anchoring and phase transition of liquid crystals to biomolecular recognition events occurring at the aqueous-LC interface. Chang-Hyun Jang is a professor in Chemistry Department, Gachon University, Seongnam, South Korea. His academic interests are liquid crystals-based biosensors, biological interactions at nanostructured interfaces, nanofabrication of proteins and particles on self-assembled monolayers, surfactant technology, and microencapsulation.