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Abstract
A new strategy is reported here to monitor the enzymatic reactions in real time by using whispering gallery mode (WGM) lasing. The optical microcavity is formed via the self-assembly of an ultraviolet (UV)-treated nematic liquid crystal (LC) 4-cyano-4’-pentylbiphenyl (5CB). The single UV-treated 5CB microdroplet serves as both optical resonator and sensing reactor. The microdroplet configuration transitions induced wavelength shift in the WGM lasing spectra can be used as an indicator for the enzymatic reaction. The proposed sensor has a sub-microgram detection limit of urease (∼0.5 µg/ml), which is lower than the detection limit of currently available urease sensor based on LC materials. Our experimental results demonstrate that WGM lasing has unique advantages in the real-time monitoring of enzymatic reactions compared, for instance, with observation of the optical appearance under a polarized optical microscope.
© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Liquid crystals (LCs) have attracted wide interest because of their ability to amplify biochemical molecular events at the aqueous/LC interface and transduce them to visible optical signals [1–3]. The overall arrangement of the LC molecules is highly sensitive to the strength and the type of local molecular anchoring at the interface so that different structural configuration of the LC can be achieved [4,5]. Using LC-based sensors, researchers have achieved the detection of biochemical molecules including proteins [6,7], glucose [8], heavy metal ions [9], artificial polymers [10–12], and lipids [4,13]. Recently, I-Hsin Lin et al [5]. used endotoxin as a functional molecule to modify LC microdroplets, of which the sensitivity can be as high as 1pg/ml. However, to date, a great number of the detector readouts (especially for LC microdroplets) are currently based on the bare-eye observation of the optical appearance under a polarized optical microscope (POM). On account of the limitations of detection method, it is difficult to effectively quantify the reaction in real time. Hereby, we propose to use whispering gallery mode (WGM) lasing in LC microdroplets for a better quantification of the reaction, which offers an alternative of solving this problem effectively.
High-quality WGM lasing can be achieved in different types of LC microdroplets due to their nearly perfect spherical structure and smooth surface [14–16]. Using WGM lasing in LC microdroplet to detect foreign molecules and enzymatic reactions carries many advantages: (i) WGM lasing can transform the configuration changes of the microdroplets into spectral information in real time. (ii) The WGM spectral shifts caused by the analytes have specific numerical values, which are more quantitative and more efficient than observing the polarized pattern of the microdroplet. (iii) The large surface-area-to-volume ratio of the LC microdroplets can ensure a high sensitivity.
Urease has a very high specificity for the catalytic hydrolysis of urea [17–20]. Normal human tissue does not contain urease and Helicobacter pylori is the only bacterium that can survive in the human stomach and secrete urease [21]. As early as 1994, the World Health Organization identified Helicobacter pylori as a Class-I carcinogen [22]. Because of its unique ability to produce a large amount of highly active urease, detecting urease is an effective method to diagnose Helicobacter pylori infections in clinical settings [23]. In previous studies, Liu et al. [19] used stearic acid doped hemispherical LC microdroplets to detect urease. The detection of microgram levels of urease was achieved using POM imaging, but there is still a need to quantitatively monitor the enzymatic hydrolysis in real-time.
We have shown in a previous study that amphipathic 4’-pentyl-biphenyl-4-carboxylic acid (PBA), as a functional material doped in 4-cyano-4’-pentylbiphenyl (5CB), can induce changes in the directional arrangement of 5CB molecules during deprotonation caused by an increasing pH [24]. However, urease detection has lower sensitivity and longer detection time using PBA because of the limited range of pH values at which PBA is reactive. Recently, Park et al. [25] reported the results of the treatment of 5CB by ultraviolet (UV) light. They found that 4-cyano-4’-biphenylcarboxylic acid (CBA) was one of the main products of photochemical degradation and its molecular structure and properties were similar to PBA. Therefore, UV-treated 5CB opens up a new possible way for the detection of urease.
Herein, we investigate the orientation properties of UV-treated of 5CB microdroplets at the aqueous/LC interface. The single UV-treated 5CB microdroplet serves as both the optical resonator and the sensing reactor. The mechanism of lasing was unambiguously identified by comprehensive spectroscopic analysis and attributed to WGMs. Urease was detected by combining WGM lasing and traditional POM imaging methods using the highly efficient and specific hydrolysis of urea. The proposed sensor has a sub-microgram detection limit of urease (∼0.5 µg/ml), which is lower than the detection limit of currently available urease sensor based on LC materials.
2. Materials and methods
2.1 Materials
5CB, urea, urease, cellulose, lipase, and acetylcholinesterase (AChE) were purchased from Sigma-Aldrich. NaCl, potassium phosphate monobasic and dibasic were used to prepare phosphate buffer solution (PBS) with different pH values and were purchased from Aladdin. 4-dicyanomethylene-2-methyl-(6-4-dimethylaminostryl)-4H-pyan (DCM) was purchased from Exciton to serve as the gain media. All aqueous solutions were prepared with deionized (DI) water purified using a Milli-Q system (Millipore, USA).
2.2 Photochemical degradation of 5CB by UV light
5CB samples were prepared using a previously published method [25]. In brief, a small beaker containing 1g of 5CB was placed under a Spectroline EN-280L longwave UV lamp (365 nm). The lamp was placed about 5 cm above the sample and enclosed in a box. The treatment time to prepare the 5CB sample was 72 hours. Based on a previous study, the concentration of the amphiphilic CBA in the 5CB samples is about 0.7% [25].
2.3 Preparation of 5CB microdroplets
First, 0.05 wt.% DCM was doped into UV-treated 5CB and mixed by ultrasonication for 30 minutes. Then, we fabricated a silica capillary tube (inner diameter: 100 µm, outer diameter: 162 µm) into a conical microtubule with a diameter of about 6 µm by using a flame-heated taper-drawing method. Next, the prepared microtubules were used to extract the previously prepared 5CB samples. Finally, by controlling the pumping rate, 5CB microdroplets with the desired size were generated in the aqueous solution. To facilitate the excitation of WGM lasing, the LC microdroplets in all experiments were generated and controlled by a homemade microtube that can generate only one microdroplet at a time. Each group of experiments was repeated at least four times to avoid artefacts.
3. Experiment and results
3.1 Effect of pH variations on configuration of UV-treated 5CB microdroplets
CBA is an amphiphilic substance containing a carboxylic acid group [25]. It is sensitive to the pH of an aqueous solution. Therefore, CBA can easily deprotonate at higher pH values [8]. We assume that the deprotonated CBA (CBA—) may self-assembles at the LC/aqueous interface to induce the orientation of the 5CB molecules from a planar [Fig. 1(a)] to a homeotropic [Fig. 1(b)] configuration. The POM images of the 5CB microdroplets will illustrate the change from the bipolar to the radial configuration [2,3].
Fig. 1. Schematic illustration of the structural transition of UV-treated 5CB microdroplet from planar alignment (a) to homeotropic alignment (b).
To demonstrate our hypothesis, the pH sensitivity of the UV-treated 5CB microdroplets was studied first. The schematic diagram for the formation of the microdroplets is shown in Fig. 2(a). The as-formed microdroplet was suspended from the end of the microtubule rather than connected to the microtubule to prevent interference from pressure changes in the microtubule. Microdroplets with the same diameter (60 µm) were generated in PBS at different pH values and the POM images were recorded after 25 minutes, as shown in Fig. 2(b). It was found that when the pH value was lower than 7, the LC microdroplets exhibited a bipolar configuration (i.e., the 5CB molecules at the interface were distributed parallel to the surface of the microdroplets). The bipolar configuration indicates that the CBA had not yet undergone deprotonation. When the pH value of the solution was 7.3, a typical transitional configuration in the form of a disclination loop [5] appeared on the surface of the LC microdroplets, indicating that the 5CB molecules gradually started changing from a planar to a homeotropic anchoring. Notably, this critical pH value for the orientation transition of 5CB induced by CBA at the plane interface was 7.5, which is close to the critical pH value at the spherical interface [8]. The reason for the slight decrease in the critical pH may be that the larger surface-area-volume ratio of the spherical structure causes more CBA molecules to be deprotonated at the interface.
Fig. 2. (a) Schematic diagram for the formation of 5CB microdroplets, SEM image of the tapered glass capillary microtube (top left inset), and micrographs of generated 5CB microdroplets in PBS (top right inset). (b) POM images of UV-treated 5CB microdroplet (60-µm-diameter) in PBS at different pH. Inset: Schematic illustrations of the director configurations for the UV-treated 5CB microdroplets in PBS at different pH.
With the increase of pH value, the disclination loop on the surface of the microdroplet became more significant, indicating that the perpendicular orientation of the 5CB molecules was more pronounced. At pH 8, the disclination loop on the surface of the LC microdroplets gradually shrank into a hedgehog-like defect point until the radial configuration was formed. The radial configuration was left unaltered for at least 25 minutes and no significant desorption of CBA— was observed.
3.2 WGM spectral response of the UV-treated 5CB microdroplets at various pH values
In principle, WGM lasing can be achieved in the optical microcavity. The excitation light of the gain dye meets the resonance condition and continuously reflects at the interface between the cavity and the surrounding medium, eventually triggering WGM lasing. Therefore, the resonant frequency of WGM lasing is very sensitive to the refractive index change in the resonator [26,27]. In our experiments, the 5CB molecules on the surface of the microdroplets were redirected due to the deprotonation of CBA. The refractive index of the microdroplet also changed accordingly. The change of the refractive index can be effectively transformed in real time into the spectral response of WGM lasing. Therefore, we attempted to use WGM lasing in LC microdroplets to quantify the enzymatic reaction better by real-time monitoring.
A schematic drawing of the setup is shown in Fig. 3(a). The pump light (wavelength: 532 nm, pulse width: 8 ns, frequency: 10 Hz) was guided to the surface of the microdroplet via a tapered optical fiber waveguide. A drop of aqueous solution was deposited on a poly(methylmetacrylate) (PMMA) substrate to serve as the host medium for the UV-treated 5CB microdroplets. A 532-nm filter was used to remove the reflection of the pump light. The emission of the microdroplet was directed with a beam splitter to a spectrometer with a 0.05 nm resolution (PG2000, Ideaoptics Technology, Ltd., China) and a charge-coupled device (CCD) camera (DP21, Olympus, Japan).
Fig. 3. (a) Schematic diagram of WGM lasing experimental setup. (b) WGM lasing spectrum of a 35-µm diameter UV-treated 5CB microdroplet in PBS (pH = 7). The calculated mode number agree with the experimental peak position. Inset: PL micrograph of lasing microdroplet, (c) PL emission of the UV-treated 5CB microdroplet with 60-µm diameter immersed in PBS (pH = 7) as a function of PPE. Scale bars, 20 µm. (d) Integrated PL intensity as a function of PPE.
To demonstrate the establishment of the WGM lasing, we first determined the lasing threshold of the UV-treated 5CB microdroplets in PBS (pH = 7). Figure 3(c) shows the photoluminescence (PL) spectra from a 60 µm UV-treated 5CB microdroplet at different pump pulse energies (PPEs). Spontaneous emission with a weak broad spectrum was observed at low PPEs (0.42 µJ). The broad spectrum changed to sharp peaks at a PPE = 0.95 µJ and their intensity increased dramatically with the PPE. A nonlinear relationship between the PL intensity and PPE can be observed [Fig. 3(d)], which indicates the presence of lasing action, and the lasing threshold was found to be about 0.86 µJ. When PPE is higher than the excitation threshold of the microdroplets, a hint of a light ring was visible around the lasing microdroplet. This can be explained with the circulating WGM lasing on the surface of the microdroplets.
The characteristics of the WGM lasing action were further studied by confirming the lasing modes number and Q factor. A 35-µm UV-treated 5CB microdroplet generated in PBS (pH = 7) was excited by pulsed lasing and the spectrum was measured by the spectrometer, as shown in Fig. 3(b). WGM lasing was clearly observed, as indicated by the sharp peaks in the spectrum. The measured lasing spectrum for the 35-µm UV-treated 5CB microdroplet in the bipolar configuration was well consistent with the first-order transverse electric (TE) modes from 288 to 294 [Fig. 3(b)], supporting the WGM lasing mechanism. The lasing modes number m can be predicted from the explicit asymptotic formulas [28]:
After determining the WGM lasing characteristics in UV-treated 5CB microdroplets, the WGM spectral response induced by pH variation was investigated on this basis. The microdroplets with 60 µm diameter were produced in PBS of pH = 7 and then the lasing spectrum was measured as shown in Fig. 4(a) (blue line). Subsequently, 40 µL NaOH solution (pH = 12) was added to PBS. The microdroplets rapidly changed from a bipolar to a radial configuration within 30 seconds. The stable WGM lasing spectrum of formed radial configuration was marked in red lines. The peak wavelength also showed a blueshift of 2.52 nm. This proved that the response of the WGM lasing to the change in the microdroplet structure can reflect the change in the environmental pH value directly. The change in the resonant spectrum can be described by [30]:
where Δn is the refractive index change in the microcavity, and Γ is the interaction factor. It has been proved that the measured WGM lasing agrees well with the TE polarization mode in this study. In the bipolar configuration, the 5CB molecules are arranged parallel to the surface. The TE polarization with the oscillating electric field has a larger dielectric constant along the long axis of the 5CB molecules that responds to the high refractive index (ne = 1.71). On the other hand, the electric field of the TM mode oscillates perpendicularly to the long axis of the 5CB molecules and experiences the ordinary refractive index (no=1.54), as shown in Fig. 4(b). Thus, the TE modes have larger index contrast to immersion liquid (n≈1.33). Upon the deprotonation of CBA, the orientation of the 5CB molecules gradually changed from parallel to perpendicular. The refractive index along the oscillation direction of the electric field decreases (no ≤ n < ne) for TE modes when the orientation of the 5CB molecules changes. Therefore, the corresponding WGM lasing spectra show a blue shift.Fig. 4. (a) WGM lasing spectra of a UV-treated 5CB microdroplet with 60-µm diameter with bipolar (blue line) and radial (red line) configurations. Schematic illustrations of the corresponding configurations within 5CB microdroplets are shown in insets (1) and (2), respectively. POM images of the corresponding configurations are shown in insets (3) and (4), respectively. Scale bars, 20 µm. (b) Schematic view of the electric field oscillation in TE (left) and TM (right) WGMs under 5CB molecules planar alignment. The yellow prolate objects represent the 5CB molecules.
3.3 Detection of urease
Urease can specifically hydrolyze urea to produce hydroxide ions and ammonium ions, which increases the local pH [19]. When the pH increases, the CBA molecules inside the UV-treated 5CB microdroplet begin to deprotonate and self-assemble at the surface of the microdroplet due to their amphiphilicity. The 5CB molecules gradually change from a planar to a homeotropic anchoring during this process (Fig. 1).
Firstly, urease was detected using POM imaging method. The microdroplets were produced in mixed pre-incubated solutions of urea (0.5 M) and urease at different concentrations (50, 5, 0.5 and 0.1 mg/ml). Figure 5(b) shows the evolution of POM images of UV-treated 5CB microdroplets in the mixture with a urease concentration of 50 µg/ml. Point defects appeared on the surface 20 seconds later as a pre-radial configuration was formed (a typical transitional configuration [5]). After 1 minute, the point defects gradually moved towards the center, showing a typical escape-radial configuration [5]. After 2 minutes, the microdroplets formed in a stable radial state. Clear cross-like patterns were observed with a hedgehog-like point defect at the center.
Fig. 5. POM images of 60-µm UV-treated 5CB microdroplets in pure PBS (pH = 7) (a), in a urea solution (0.5M) (e), and in a urease solution (50 µg/ml) (f). Evolution of the POM images of 60-µm UV-treated 5CB microdroplets in pre-incubated mixed solution of 0.5M urea and 50 µg/ml (b), 5 µg/ml (c), 0.5 µg/ml (d) urease. Scale bars, 20 µm.
In order to preliminarily determine the limit of detection for urease, the concentration of urease in the mixture was reduced to 5 µg/ml, as shown in Fig. 5(c). The LC microdroplets were in a good radial configuration within 6 minutes. Unlike the transitional configuration in the high-concentration urease solution, a significant disclination loop was observed. For a urease concentration of 0.5 µg/ml, the radial configuration was not formed [Fig. 5(d)], possibly because the concentration of hydroxide ions produced by the reaction was too low to force enough CBA molecules to deprotonate.
Furthermore, to verify that the deprotonated CBA was produced by urease hydrolysis of urea, we generated UV-treated 5CB microdroplets with the same parameter configuration in pure PBS (pH = 7), in a urea solution (0.5 M) without urease, and in a urease solution (50 µg/ml) without urea. The microdroplets in all three solutions had a bipolar configuration that remained unchanged for at least 25 minutes [Figs. 5(a), 5(e), and 5(f)]. These results indicate that the deprotonation of CBA is indeed due to the enzymatic hydrolysis of urea in the experiment.
Next, we investigated the WGM spectral response of the UV-treated 5CB microdroplets in solutions with different urease concentrations. The spectra were repeatedly recorded at specific times. To avoid degradation of the dye molecules, the microdroplets were only excited for a short period (about 1 second). Figure 6(a) shows the wavelength shift in the WGM lasing spectra for various concentrations of urease. The concentration of the enzyme has a significant effect on the intensity and the duration for the hydrolysis reaction to complete. Generally, higher enzyme concentrations produce a more intense hydrolysis reaction within a certain range of concentrations. Our experimental results supported this known fact since the shift of the lasing spectra decreased when urease concentration decreased, and the reaction took longer to reach an equilibrium.
Fig. 6. (a) Temporal dependence of WGM wavelength shift for different concentrations of urease. (b) Portion of WGM lasing spectra as a function of time. The spectra were collected from a 60-µm-diameter UV-treated 5CB microdroplet in pre-incubated solution of 0.5M urea and 0.5 µg/ml urease.
Notably, for urea concentrations of 50 µg/ml and 5 µg/ml, the UV-treated 5CB microdroplets were in the same radial state but yielded different spectral shifts. This indicated that more information could be obtained by using the WGM spectral response to monitor the reaction process than by simply observing POM images. The detection limit of urease was at the sub-microgram level (∼0.5 µg/ml). At this concentration, the WGM spectra showed a blue shift of 1.04 nm within 14 minutes with good linearity [Fig. 6(b)]. Moreover, we demonstrated that for various concentration of urease, the starting time and the range of wavelength shift are quite different and have specific numerical values. This means that the detection of enzymatic reaction can be transformed into effective and quantitative spectral shift information. In this regards, our microdroplet-based sensing or detecting system has a preferable performance.
3.4 Selectivity of the urease sensor
The optical responses of UV-treated 5CB microdroplets in pre-incubated mixtures of urea and other enzymes, such as pectinase, cellulase, lipase, and acetylcholinesterase (AChE), also were studied to demonstrate the high selectivity of our urease sensor. In the detection of the four selected enzymes, the configuration of all 5CB microdroplets exhibited a bipolar configuration [Figs. 7(a2)–(a5)].
The WGM lasing was also used to test the selectivity of the urease sensor. Within 25 minutes, no significant spectral shift was observed in any experimental group except in the one with urease [ Fig. 7(b)]. This demonstrated that the sensing system had a good selectivity for urease and low interference. In addition, the lasing spectra at the 25th minute of microdroplets with the same diameter generated in different solutions are shown in Fig. 7(c). The position of the WGM lasing peaks only changed within 0.4 nm except when urease was present. We determined that the refractive index of all tested solutions in the range from 1.33 to 1.34, thereby ruling out effects from the optical properties of the medium. This indicates that the lasing behavior of LC microdroplets depends more on the arrangement states of LC molecules when the external refractive index changes little.
Fig. 7. (a) POM images (recorded after 25 min) of 60-µm-diameter UV-treated 5CB microdroplets in DI water (1), pre-incubated solution of 0.5M urea and 50 µg/ml pectinase (2), 50 µg/ml cellulose (3), 50 µg/ml lipase (4), 50 µg/ml AChE (5), and 5 µg/ml urease (6). Scale bars, 20 µm. (b) WGM lasing wavelength shift of UV-treated 5CB microdroplets in different solutions. (c) WGM lasing spectra of UV-treated 5CB microdroplets in different solutions. The spectra were recorded at the 25th min after the formation of microdroplets.
4. Conclusion
In conclusion, we have developed a new strategy for real-time monitoring of urease by using the WGM spectral response. A single UV-treated 5CB microdroplet acted as the optical resonator and the sensing reactor. CBA was produced by photodegradation of 5CB, and no additional functional materials (e.g., PBA or stearic acid) doping was required. Deprotonation of CBA induced the transition of the anchoring direction of the 5CB molecules, resulting in changes in the spectral response of the WGM lasing and in the POM images. We compared both methods in our studies, and the results demonstrated that urease can be quantitatively and specifically monitored in real time by using WGM lasing technology. The proposed sensor has a detection limit of urease as low as sub-microgram level. These results indicated that this novel strategy holds great promise for application in the real-time monitoring of pH-related biochemical reactions and can be expected to replace conventional POM observations for current LC-based biosensor systems.
Funding
National Key R&D Program of China (2016YFF0200704, 2017YFB0405502); National Natural Science Foundation of China (61635007).
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