Label-free, color-indicating, and sensitive biosensors of cholesteric liquid crystals on a single vertically aligned substrate

: Biosensors based on liquid crystal (LC) materials can be made by employing the sensitive interfacial eﬀect between LC molecules and alignment layers on substrates. In the past, the optical texture observation method was used in the LC biosensor ﬁeld. However, the method is limited by a complicated fabrication process and quantitative reproducibility of results that bv evidence that both the reliability and accuracy of LC biosensors need to be improved. In this report, we demonstrate that cholesteric LC (CLC) cells in which one substrate is coated with a vertically aligned layer can be used as a new sensing technology. The chirality of the single vertically anchored (SVA)/CLC biosensor was tested by detecting bovine serum albumin (BSA), a protein standard commonly used in the lab. The colors and corresponding spectrum of the SVA/CLC biosensor changed with the BSA concentrations. A detection limit of 1 ng/ml was observed for the SVA/CLC biosensor. The linear optical properties of the SVA/CLC biosensor produced cheap, inexpensive, and color-indicating detection of biomolecules, and may promote the technology of point-of-care devices for disease-related biomarker detection.


Introduction
The use of a nematic liquid crystal (LC) to detect biomolecules was first proposed by Abbott's group [1]. Since then, LC biosensors have received the attention of scientists [2][3][4]. The mechanism of LC biosensors is that biomolecules immobilized on substrate surfaces induce the vertical-to-planar reorientation of LC molecules [4]. Thus, dark-to-bright optical responses of LCs can be observed under a microscope with crossed polarizers [5]. In addition, the reorientation of LC molecules interacting with biomolecules can be described by the molecular theory of surface tension [6]. Now, LC-based sensing techniques are used to detect enzymatic reactions, antibody-antigen immunoreactions, and DNA [1,3,4]. In addition, recent studies have show that LC immunodetection of the cancer biomarker CA125 can be achieved using both a typical nematic LC (5CB) and an LC with large birefringence (∆n = 0.333 at 589 nm and 20°C), [7][8][9]. However, present LC-based biosensing techniques are limited by optical texture observations. The non-uniformity of LC texture images are difficult to reproduce and quantitate.
Cholesteric LCs (CLCs) are a special material which possess unique properties such as Bragg reflection, bistability, and flexibility [10,11]. Furthermore, the helical structure of CLCs selectively reflects light with the same handedness as the CLC chirality. In addition, both the planar (P) and focal conic (FC) states are bistable, meaning that no voltage is needed to maintain the two optical states of CLCs [12]. Based on the properties of CLCs, many applications have been proposed, such as tunable photonic crystal devices [13][14][15], displays [16], and optic devices [17,18]. Despite their photonic applications, the potential of CLCs as biosensors have rarely been investigated. Hsiao et al. proposed the first CLC biosensor device in 2015 [19], and a highly sensitive color-indicating quantitative biosensor was presented. It is a pity that CLC-based biosensors in the literature require complicated fabrication processes. In this report, a simple special CLC biosensor on a single vertically aligned substrate is demonstrated. The highly sensitive interface between CLC molecules and a single vertical anchor (SVA) consisting of N,N-dimethyl-n-octadecyl-3-aminopropyltrimethoxysilyl chloride (DMOAP) was used to detect concentrations of a common protein standard, bovine serum albumin (BSA). A schematic of this SVA/CLC biosensor is shown in Fig. 1. We know that CLC molecules are vertically aligned near the substrate by DMOAP. The non-coated alignment layer of the glass substrate also causes CLC molecules to be in disarray. CLC molecules are in the hybrid two bulk of the FC state and the one bulk of the P state through self-assembly in the intermediate space away from the DMOAP or glass substrate, leading to the configuration of a CLC device with a major reflection mode. Moreover, the vertical anchoring strength of CLC molecules is weakened when the biomolecules are adsorbed onto DMOAP-coated substrates. These biomolecules allow CLC molecules to transfer to the P structure on the DMOAP-coated side which causes the device to be in the major transmission mode. In addition, the transition of the CLC structure from major reflection to major transmission mode brings about the color-indicating properties of SVA/CLC Here we neglected the top substrate, which was also coated with DMOAP, because the air also induced focal conic state of CLC as DMOAP did.
devices. Quantitation of the concentrations of BSA with a lower detection limit of ∼1 ng/ml can be realized by measuring the SVA/CLC biosensor using transmission spectroscopy.

Experiment
The CLCs used in this study were a nematic LC E7 as the host and the chiral dopant, R5011. The concentration of R5011 was 2.6 wt%, and the pitch of the CLC was located at near 590 nm. The glass was immersed in a 1% DMOAP aqueous solution for 15 min at room temperature to coat the vertically aligned layer onto the substrate, and then it was rinsed with deionized (DI) water for 1 min to remove any excess DMOAP solution on the substrate. In addition, BSA immobilization was fulfilled by the DMOAP-coated glass in aqueous solutions of BSA at concentrations of 1 mg/ml, 1 µg/ml, and 1 ng/ml, respectively. To fabricate an empty SVA cell, spacers mixed with ethanol were distributed on the DMOAP-or BSA-coated slides, which were covered with another glass slide. Finally, the empty cell was filled with CLC by capillary action to form an SVA/CLC cell. The thickness of the SVA/CLC biosensor is about 12 µm. The soaking time of BSA immobilization is about 30 min. A polarized optical microscope (POM) was used in this study to observe the texture. Transmission spectra of SVA/CLC cells were measured with a high-resolution fiber-optic spectrometer (Ocean Optics. HR2000+).

Results and discussion
The optical textures of four SVA/CLC biosensors in various concentrations of BSA are shown in Fig. 2. One can observe that the brightness of the optical texture increased with an increasing concentration of BSA. Because the light scattering caused by the random CLC structure was much stronger at lower BSA concentrations and the number of defect lines in the optical textures were reduced when the BSA concentration increased, the content of the random focal conic (FC) state was higher in the non-biomolecule situation. Note that the chiral dopant material, R5011, used in this study was temperature-independent [20]. Thus, the SVA/CLC biosensor can be conveniently and consistently used in a wide range of room temperatures in different countries. The chiral pitch of the CLC is not thermosensitive, which is advantageous for various biosensor applications. SVA/CLC biosensors with immobilized BSA at 0∼1 mg/ml are shown in Fig. 3. Different colors were reflected from the SVA/CLC biosensor at each BSA concentration. In the absence of BSA, the reflected color of CLC was orange (major reflection mode), which shifted to yellow, green, and blue with increasing concentrations of BSA. Thus, it is possible to determine the amount of biomolecules on a logarithmic scale by observing color-indications of SVA/CLCs. The different reflected colors from the SVA/CLC biosensors depend on the structure of CLC, which is converted from the FC-P-FC state to the FC-P state by interacting with BSA and CLC molecules as shown in Fig. 3. In addition, SVA/CLC biosensor was in the major-reflection mode when immobilized in the absence of BSA. The light passing through the upper substrate of the SVC cell was reflected by the partial FC state of the CLCs. The function of colors change-dependent concentrations of BSA is resulted from the arrangement change of CLC molecules. The transmittance of blue light (complementary color of orange light) through the P state was scattered by the FC layer onto the lower substrate, causing the color orange to appear from the SVA/CLC biosensor. However, the vertical anchoring force of the aligned DMOAP layer was diminished by the immobilized BSA. CLC molecules converted to the P state with increasing concentrations of BSA. Finally, the perfect P state caused the SVA/CLC biosensors to be in major transmission mode (Fig. 4), and the appearance of the device became the complementary color of blue on the white paper underneath the SVA/CLC biosensor (Fig. 3). In the configuration of the sample demonstrated in this work, one substrate is BSA-modified while the other one is DMOAP coated. The DMOAP coated substrate can homeotropically align liquid crystal molecules and induce focal conic phase for CLC. However, air can homeotropically align liquid crystal molecules and induce focal conic phase for CLC as well. Therefore, it is anticipated that the DMOAP coated substrate can be removed from this sample but remaining the function of sensing. In order to achieve a quantitative method for SVA/CLC biosensors, the transmission spectra of SVA/CLC devices immobilized with various concentrations of BSA are shown in Fig. 5. The transmittance of SVA/CLC biosensors was raised, and the reflection bandwidth decreased with an increasing BSA concentration. We determined that the limit of detection of the SVA/CLC biosensor was 1 ng/ml of BSA. The CLC in the vicinity of the lower glass substrate was in the FC state at low BSA concentrations. Thus, light scattering undermined Bragg reflection. When the BSA concentration increased, the P state of CLC was predominant in the lower glass substrate. Thus, the optical response of the SVA was governed by Bragg reflection. The photonic band gap was more integrated with an increasing BSA concentration. In addition, both the minimum transmittance and bandwidth at half maximum of the Bragg reflection are shown against BSA concentrations. Based on these properties, the logarithmic scale biosensor as positive and negative correlation methods was demonstrated (see Fig. 6 and Fig. 7). Note that the linear correlation between the transmittance of Bragg reflection of SVA/CLC and different BSA concentrations is shown in Fig. 7. These results proved that the Bragg reflection of CLCs in the spectrum can be used for detecting and quantitating biomolecules in linear. Furthermore, grating coupled interferometry (GCI) and plasmonic sensors are label-free biosensing technique [21][22][23][24][25][26]. Compared to GCI and plasmonic sensors, our label-free SVA/CLC biosensor is cheaper and can be observed by the naked eyes, based on the color indications and ease of manufacture. This study demonstrates that CLCs have potential in the development as a sensitive, color-indicating, cheap, and simply fabricated biosensing technique.

Conclusions
We have successfully presented a novel SVA/CLC biosensing technique for detecting and quantitating biomolecules. The chirality of the vertically aligned CLC molecules was sensitive to the amount BSA on the alignment layer. The reflection and transmission of light leading to the color indication of the SVA/CLC biosensor could be observed. In addition, a quantitative method on a logarithmic scale correlated with BSA concentrations was also demonstrated with the spectral properties of CLC. The detection limit of the SVA/CLC biosensor was about 1 ng/ml, which is the same performance as conventional methods based on textural observations. The SVA/CLC biosensor was shown to be a simple and inexpensive platform for sensitive and color-indicating detection of biomolecules. It may facilitate the development of proposed devices for detecting disease-related biomarkers. This paper is a significant step toward the practical application for clinical use since it proves that a single vertically aligned substrate can also effectively sense BSA concentrations.