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Liquid crystal-based aptamer sensor for sensitive detection of bisphenol A
Microchemical Journal 146 (2019) 1064–1071
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Liquid crystal-based aptamer sensor for sensitive detection of bisphenol A Huihui Ren, Zongfu An, Chang-Hyun Jang
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Department of Chemistry, Gachon University, Seongnam-daero1342, Sujeong-gu, Seongnam-si, Gyeonggi-do 13120, Republic of Korea
ARTICLE INFO
ABSTRACT
Keywords: Aptamer sensor Liquid crystal 4-Cyano-4′-pentybiphenyl Bisphenol A
Bisphenol A (BPA) is an environmental poison that seriously affects the health of humans. Herein, a novel, labelfree, time-saving liquid crystal-based aptamer sensor for the detection of BPA was developed based on the specific binding of an aptamer and its target. In this study, the surface of the sensor was assembled with (3aminopropyl)triethoxysilane & dimethyloctadecyl[3-(trimethoxysilyl) propyl]ammonium chloride and the amino-aptamer was attached to this film via glutaradehyde links. Through a specific reaction, BPA reacts with the BPA aptamer forming an ‘aptamer–BPA’ complex that changes the conformation of the BPA aptamer. Through spatial size effects, the large size of the complex disturbs the nematic orientation of 4-cyano-4′-pentybiphenyl in the film on this surface from homeotropic to random, resulting in an image that changes from ‘dark’ to ‘bright’ under a polarized light microscope. The limit of detection of this system is 600 pM. Atomic force microscopy was also employed to further validate the results.
1. Introduction Bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl)proane, C15H16O2) is an endocrine disrupting chemical bearing two phenol functional groups (Fig. 1A) that is considered an environmental hormone substance [1]. BPA has always drawn great concern because it is one of the main raw and processed materials in the manufacture of epoxy resins and polycarbonate (PC) [2]. It has also other applications such as in baby supplies, food-grade packaging materials, and pesticides [3,4]. It has been reported that BPA is released into the environment mainly through sewage effluents by leaching from the epoxy resin and commercial PC products during the corresponding industrial processes [5,6]. More importantly, many studies have shown that BPA has great impact on the human body related to damage of the reproductive function and other diseases [7,8], such as miscarriages, precocious puberty, prostate growth, cancer [9], being introduced in the human body via the food chain and enriched by bioaccumulation [10]. In addition, its excretion is difficult because BPA is not easily degraded [11]. Although the level of BPA in the environment is extremely low, it still causes blockage of the neural system, malformation and canceration of organs, functional disorders of the endocrine-immune system, and interference with the enzymatic system [12]. According to the public Health Service Food and Drug Administration (FDA), the tolerable daily intake value (TDI) is 5 mg/kg bw/day (23.126 μM) [13]. Thus, the development of rapid, sensitive, and simple methods to determine BPA levels is urgent. Various analytical methods have been developed to detect BPA,
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which include: gas chromatography–mass spectrometry (GC–MS) [14], liquid chromatography-mass spectrometry (LC-MS) [15], high-performance liquid chromatography (HPLC) [16], Enzyme-Linked Immunosorbent Assay (ELISA) [17], electrochemical technologies [18], and molecular imprinting methods [19]. However, most of these approaches need long times, expensive and complex instrumentation, or a specific labeling agent, and are thus not suitable for the detection of BPA. Therefore, the development of low-cost, efficient, high-sensitivity, fast, and label-free methods remains a big challenge. We have developed a liquid crystal-based aptamer sensor. It has a limit of detection (LOD) of 600 pM, and a linear range from 20 to 600 pM and from 600 pM to 30 nM. In order to illustrated the sensor performance, the characteristics of LC-based aptamer sensor were compared to other reported methods in Table S1. Ye et al. developed an electrochemiluminescence aptamer sensor for BPA detection in milk and water. The device used an Au electrode, which achieved a linear range from 0.1 to 100 pM and an LOD of 76 fM with detection time of 7 h [20]. Yu et al. presented electrochemical aptamer sensor using triple-signaling strategy to detect BPA in real-water samples in approximately 3 h. This sensor had an LOD of 0.19 pM with a linear range from 1 to 100 pM [21]. Kazane et al. reported electrochemical aptamer sensor based on Poly (Pyrrole-Nitrilotriacetic Acid)-Aptamer Film to detect BPA with detection time of 30 min. The device had a linear range from 10 pM to 1 μM with an LOD of 10 pM [22]. Zhu et al. developed a fluorescence aptamer sensor for detecting BPA in actual water samples. The device used a surface of graphene oxide and detect BPA from 0.1 to
Corresponding author. E-mail address: [email protected] (C.-H. Jang).
https://doi.org/10.1016/j.microc.2019.02.019 Received 27 September 2018; Received in revised form 30 January 2019; Accepted 7 February 2019 Available online 08 February 2019 0026-265X/ © 2019 Published by Elsevier B.V.
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phenomenon was found to be reversible [32]. Later, Lehmann also observed this phenomenon and found that the mesophase (the turbid liquid) presented similar properties to those of a crystal [33]. Thus, a liquid that presents fluidity and optical anisotropy is called an LC. The principle of LC-based biosensors relies on the unparalleled conformation of the LC line phases and the elastic, optical, anchoring, and birefringent properties of the LC material [34]. LC material has the property of birefringence that could be observed by polarizing microscope. There are two polarizing plates perpendicular to each other (the lower polarizer is called a polarizer and the upper polarizer is regarded as an analyzer). When natural light passes through the polarizer, it becomes linearly polarized light. As showed in Fig. 1B, when LCs are vertically aligned and the linearly polarized light passes through the LCs, the vibration direction of polarized light and the analyzer is perpendicular to each other. Because the vibration direction of the polarized light cannot be changed, the observed field is black. If the LCs are planar, there is a great change in the vibration direction of polarized light under the influence of LCs. At this time, part of the polarized light can go through the analyzer, so the brightness images could be obtained. Incorporation of receptors and ligands on the surface of the biosensor can disrupt the alignment of LC molecules [35]. This deviation can then be processed optically, and the arrangement of LCs can be correlated with the identity and/or concentration of the analyte. Most of these LC-based biosensors depend on the binding of biological macromolecules that change the alignment of the LCs on the surface. However, to date, only a few LC-based sensors have been proposed that can be used to detect small organic molecules [36]. This study reports a highly sensitive method to detect BPA using an LC-based aptamer sensor. Biosensors with biomolecules as sensing elements have become the most promising method for BPA detection because of their high response rate, high specificity, and simple operation [37]. Recently, antibodies have been extensively applied to discriminate biological elements; however, due to their lengthy preparation, and great instability, their application in biosensors remains limited [38]. Compared to traditional approaches based on antibodies, new recognition elements for aptamers [39], such as single-stranded DNA or RNA (ssDNA or ssRNA, respectively), can be screened from large mononucleotide databases through the Systemic Evolution of Ligands by Exponential Enrichment (SELEX) technique [40]. In addition, aptamers can selectively bind proteins [41], small organic molecules [42], antibodies [43], and even cells [44]. Recently, more and more recognition elements have led to the fabrication of biosensors, a field where aptamers have been gaining significance. In addition to highaffinity binding and high-specificity recognition characteristics, similar to those of antibodies, aptamers also exhibit superior properties such as low molecular weight, wide range of target molecules, easy repetitive synthesis and labeling, stability during storage, and susceptibility toward denaturation [45].
Fig. 1. (A) Chemical structure of BPA; (B) Schematic illustration of optical switching behaviors of 5CB.
10 ng/mL with an LOD of 0.05 ng/mL [23]. Chen et al. demonstrated a fluorescence aptamer sensor fabricated by construction of the DNA Y junction. The device could detect BPA concentration as low as 5 fM with detection time of 90 min [24]. Mirzajani et al. designed a PCBbased capacitive aptamer sensor for label-free analysis of BPA in canned foods with detection time of 20 s. The sensor could detect BPA from 1 fM to 10 pM with an LOD of 152.93 aM [25]. When compare to other techniques, our LC-based sensing approach possesses a range of characteristics that make it worthy of exploration. These characteristics include high speed, simple operation, low cost, and high sensitivity. Generation of optical outputs can be easily detected and quantified using visible light. It does not require laborious labeling, fluorescent or redox tags, or the need for complex instrumentation or highly skilled personnel. As for detection time, although the reaction between aptamer and BPA takes 1 h, amplification (reorientation of LC) can occur within a second. The amplification step does not need to be carefully timed, as in case of ELISA, thus simplifying the design of multiplexed analyses. Liquid crystals (LCs) are substances between the crystalline solid state and isotropic liquid state [26] that exhibit optical anisotropy and long-range orientation [27], and that can also be used to magnify and convert chemical signals into electrical signals [28]. These properties, coupled with their rapid response to external stimuli, render LCs very suitable as “sensing elements” [29]. The phenomenon of liquid crystallinity was discovered by Friedrich Reinitzer, a famous botanist, while studying cholesteryl benzoate (C6H5CO2C27H48, CB) [30] in the 1980s [31]. At that time, Reinitzer found that there were two distinct melting points in cholesteryl benzoate. At a temperature of 145.5 °C, the compound melts into a turbid liquid state but, when heated to 178.5 °C, the cloudy liquid changes into a transparent, isotropic liquid; this
2. Materials and methods 2.1. Materials (3-Aminopropyl)triethoxysilane (APTES), dimethyloctadecyl[3-(trimethoxysilyl) propyl]ammonium chloride (DMOAP), bisphenol A, bisphenol B (BPB), and bisphenol C (BPC) were purchased from SigmaAldrich (St. Louis, MI, USA). Hydrogen peroxide (30% w/v), methanol, ethanol (anhydrous), sulfuric acid, phosphate-buffered saline (PBS), Tris-buffered saline (TBS), and glutaradehyde (GA) were purchased from Daejung Chemicvals & Metals Co. Ltd., South Korea. The aptamer used in the experiments (5′/5AmMC6/CCG CCG TTG GTG TGG TGG GCC TAG GGC CGG CGG CGC ACA GCT GTT ATA GAC GTC TCC AGC3′) was synthesized by Integrated DNA Technologies. Nematic LCs of 4cyano-4′-pentylbiphenyl (5CB) were obtained from Tokyo Chemical Industry Co., Ltd. (Japan). Micro slide glasses were purchased from Fisher Scientific (Pittsburgh, PA). All aqueous solutions were prepared 1065
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with deionized (DI) water (18.2 MΩ cm−1) using a Milli-Q water purification system (Millipore; Bedford, MA).
about 1.5 cm × 1 cm. A DMOAP-coated glass slide was used as the top surface, with an APTES/DMOAP-coated glass slide as the bottom surface placed face to face. The two surfaces were spaced by two thin polyester films (Mylar, 19 μm). The reaction proceeded on the functionalized APTES/DMOAP-coated surface, where 5CB was introduced in the cells under heating (at approximately 60 °C). Subsequently, the optical cells were slowly cooled and then polarized light microscopy was used to analyze them.
2.2. Cleaning of substances The micro slide glasses were immersed in a newly prepared piranha solution (v(H2SO4)/v(H2O2) = 7:3) for 30 min at 85 °C to remove all organic contaminants under nitrogen atmosphere (0.2 MPa). After that, they were rinsed three times with DI water, ethanol, and methanol, dried under nitrogen flow, and stored in an oven at 110 °C overnight prior to being decorated with APTES/DMOAP.
2.8. Atomic force microscope (AFM) measurements The root mean square roughness (RMS) and morphology of the modified mixed APTES/DMOAP self-assembled single layer were characterized via AFM (NanoScope IIIa, Veeco Metrology, Santa Barbara, CA). The AFM images were captured at a scan rate of 1.0 Hz and scanning area of 1 μm × 1 μm.
2.3. DMOAP decoration The washed micro slide glasses were immersed in an aqueous solution containing 0.4% (v/v) DMOAP for 30 min at room temperature, and then rinsed with large amounts of DI water. The DMOAP-decorated slide glasses were dried under a stream of nitrogen gas and stored in an oven for 1 h at 110 °C.
2.9. Optical image acquisition by polarized light microscopy (POM) All images of the LC optical response were taken using a polarized light microscope (Eclipse LV100 POL; Nikon; Tokyo, Japan) equipped with a digital camera (DS-2Mv; Nikon; Tokyo, Japan). All the optical images were obtained using a 4× objective lens between the crossed polarizer and the analyzer at a resolution of 1600 × 1200 pixels, gain of 1.00×, and shutter speed of 1/10 s.
2.4. APTES/DMOAP decoration The cleaned glass slides were immersed in an ethanol solution containing 1% (v/v) DMOAP and 5% (v/v) APTES for 1 h at 80 °C, and then successively rinsed with a large excess of ethanol and DI water to remove any physically absorbed APTES/DMOAP. Next, the slides were dried under a stream of N2 and stored in an oven for 1 h at 110 °C. Finally, the slides were immersed in a 1% (v/v) GA aqueous solution for 30 min, rinsed with DI water, and dried under nitrogen flow.
3. Results and discussion 3.1. BPA detection method
2.5. BPA-aptamer immobilization
The detection mechanism of the present LC sensor is shown in Fig. 2, based on the high specificity of the aptamer and its target molecules. The aptamer configuration was labeled with an amino group (eNH2) at the 3′-end of the chain, which was used to form a stable amide bond with the aldehyde group of GA, so that the aptamer could be fixed on the substrate. The sensing film was constructed with two reagents (APTES and DMOAP) to provide a long chain favoring the vertical alignment of LC molecules and an active group for biomolecule immobilization. Upon coating with GA (Fig. 2B), the amino-aptamer was fixed on the APTES/ DMOAP-modified glass slides (Fig. 2C). Due to the specificity of the aptamer and its target molecule (BPA), BPA can be effectively captured on the aptamer-based surface. In this context, the BPA aptamer serves as an identification element: when it reacts with BPA, its conformation changes and an ‘aptamer–BPA’ complex is formed (Fig. 2D). The large size of the complex greatly increases the density at the bottom film, remarkably disturbing the alignment of LCs so that 5CB molecules undergo an orientation transition from homeotropic to random under these conditions (Fig. 2F). Owing to the birefringence effect of the LC molecules, changes in the brightness of the image are observed by POM and rapid detection of BPA is realized. In the absence of BPA molecules, the conformation of the aptamer remains in the original state (Fig. 2E). In this case, the aptamer as an interface is insufficient to trigger a random orientation profile and thus the optical response observed by POM reveals a uniform black image.
The BPA aptamer was immobilized on the glass slides using a GA cross-linking method. First, the BPA aptamer was dissolved in 10 mM PBS (pH = 7.4, 2.7 mM KCl, 137 mM NaCl) and stored at 4 °C for use. After that, the GA-activated glass slides were immersed in the prepared aptamer solution at room temperature for 1 h, then rinsed successively with PBS buffer and DI water, dried under a stream of N2, and finally stored in an oven for 1 h at 110 °C. 2.6. Specific binding between the BPA aptamer and BPA The BPA aptamer-bound glass slides were treated with 80 mM glycine in aqueous solution for 1 h in order to block the unreacted aldehyde binding sites on the glass slides. Afterwards, BPA solutions at different concentrations were dropped on the BPA aptamer-immobilized glass slides at room temperature for 1 h, then rinsed with 50 mM TBS buffer (pH = 8.0) and DI water to remove any physically bound molecules, and finally dried with N2 gas to produce the final platform. In order to verify the practicality of the developed sensor, real samples containing BPA (plastic products such as a CD disc and plastic bag) were tested. In short, 1.0 g of each plastic product was placed in 15 mL DI water and processed by ultrasonication for 1 h at 90 °C; after that, the samples were stirred at 90 °C for 48 h and finally filtered. The final samples were stored in a refrigerator at 4 °C. Orange juice doped with BPA was also analyzed. The orange juice sample was centrifuge at 3000g for 30 min to remove any particle, and then diluted with 10 mM PBS (pH 7.4) at the ratio of 1:1. We added BPA into the sample to obtain a 20 μM solution of BPA. BPA was then measured by the method described above. Each experiment was repeated three times.
3.2. Optimization of fundamental conditions The orientation of LCs on the DMOAP-modified surface can be influenced by APTES. We therefore investigated the effect of the self-assembly of APTES/DMOAP on the orientational response upon incubation in APTES/DMOAP mixtures at different ratios. As shown in Fig. S1, the darkness of the optical LC image decreased with the gradual increase of the APTES/DMOAP volume ratio until a bright image was obtained. When the proportion of APTES/DMOAP was 1:1, a completely dark stabilized background was observed, corresponding to a
2.7. Preparation of sandwich LC optical cells The optical cells were constructed with two different solid surfaces and the LCs sandwiched in-between. The size of the optical cells was 1066
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Fig. 2. Diagram of the LC sensing device for BPA detection: (A) clean glass slide, (B) immobilization of (APTES/GA) DMOAP for 30 min at room temperature on the glass, (C) immobilization of the BPA aptamer for 1 h at room temperature, (D) incubation with BPA for 1 h at room temperature, (E) vertical alignment of 5CB in the optical cell in the absence of BPA, and (F) random orientation of 5CB after treatment with BPA. POM photographs are also shown.
perpendicular orientation of the LC molecules (Fig. S1A). The results demonstrated that the presence of small amounts of APTES does not perturb the perpendicular alignment of LCs in the film. In Fig. S1B, a dark texture with some bright spots is observed for the APTES/DMOAP solution at 5:1 ratio. In addition, compared to Fig. S1A and B, many bright spots are observed at an APTES/DMOAP volume ratio of 10:1 (Fig. S1C). At a higher ratio of APTES/DMOAP (20:1), the number of bright spots in the POM image increases, almost covering the whole image (Fig. S1D). In order to obtain enough signal-to-background contrast and good self-assembly efficiency for BPA-aptamer anchoring, an APTES/DMOAP proportion of 5:1 was finally chosen to immobilize GA. The signal background is also influenced by the amount of GA activating the APTES/DMOAP self-assembled film. Optimization experiments were conducted at various concentrations of GA and the results are shown in Fig. S2. It was found that, at a concentration of GA (v/v) of 0.5% and 1% (Fig. S2A and B), the optical appearance of LC cells showed a uniform black image. In contrast, when the concentration of GA (v/v) reached 4% (Fig. S2C), the liquid crystal arrangement was disturbed and a bright texture appeared in the POM images. In order to ensure a sufficient number of aldehyde groups for immobilization of the BPA aptamer and weak background signal interference, 1% GA was employed in subsequent experiments. It is well known that the orientational profile of 5CB molecules can
also be influenced by the surface of aptamers coated on glass slides. In order to improve the signal-to-background contrast and obtain a zerointerference background, experiments were conducted to optimize the concentration of BPA aptamer. As seen in Fig. S3, the extent of colored areas in the optical image decreased with the concentration of BPA aptamer. In Fig. S3A and B, at low aptamer concentrations (100 nM and 1 μM), uniformly black optical appearance was observed, suggesting that such loadings of BPA aptamer molecules are unable to influence the arrangement of LC molecules, and that higher concentrations are needed to ensure that enough aptamer molecules are present to obtain adequate BPA detection. When the concentration of aptamer was increased to 5 μM and 10 μM, multiple bright colored areas were observed (Fig. S3C and D). A large number of BPA aptamers were immobilized on the LC-based sensor, which had a great influence on the alignment of the 5CB molecules, causing large changes in the intensity and brightness of the optical image. Upon further increasing the concentration of aptamer, where the LC random arrangement was maintained, a greatly colored texture was observed in the optical image. Therefore, a concentration of BPA aptamer of 1 μM was chosen to provide adequate sensitivity of the subsequent self-assembly. In summary, the optimal conditions for the alignment of liquid crystal molecules were determined to be an APTES/DMOAP ratio of 5:1; 1% GA to bind the BPA aptamer, and a minimum concentration of 1067
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Fig. 3. POM images of LC sensing cells sandwiched between an upper surface immobilized 0.4% DMOAP and a lower surface exposed to various concentration of BPA: (A) 20 pM; (B) 200 pM; (C) 600 pM; (D) 2 nM; (E) 20 nM; (F) 200 nM; (G) 2 μM and (H) 20 μM in TBS. Scale bar: 100 μm.
(R2 = 0.9807) and Grayscale = 54.377 logCBPA-139.89 (R2 = 0.9952) respectively. CBPA means the concentration of BPA. Based on the obtained image, a gradation analysis of the images can be performed and therefore, the quantitative analysis of BPA can be realized within a certain concentration range.
BPA aptamer of 1 μM to ensure the sensitivity of the aptamer sensor through the rearrangement of the liquid crystal alignment from homeotropic to random. 3.3. Limit of detection
3.4. Selectivity
In view of the fact that the anisotropic surface topography of the substrate is significantly affected by BPA, a study of the specific interaction between BPA and the BPA aptamer by cross-polarized optical imaging was performed. The configuration of the BPA aptamer, as the specific recognition element of the LC-based sensor, changes upon binding BPA. According to these results, we hypothesized that the alignment of 5CB molecules would be perturbed by the formation of a ‘BPA–aptamer’ complex via hydrogen bonding and ion pairing. Hence, a series of concentrations of BPA were tested under this assumption and optical images were obtained with a polarized light microscope. As shown in Fig. 3, the intensity and region of defects gradually increased to yield a completely bright texture with the increasing BPA concentration. A fully black optical appearance was obtained at BPA concentrations of 20 and 200 pM (Fig. 3A and B), showing that such BPA loadings are unable to perturb the perpendicular orientation of the surface. In Fig. 3C, at a BPA concentration of 600 pM, some obvious bright spots in the optical images were detected compared to Fig. 3A and B. In contrast, the optical response of LC cells displayed apparent brightness at BPA concentrations of 2 nM, 20 nM, 200 nM, 2 μM, and 20 μM (Fig. 3D, E, F, G and H respectively). This is due to high concentrations of BPA being able to disrupt the alignment of 5CB molecules, making the LC optical image bright. These results are consistent with our assumption that specific binding of BPA molecules and the surface-immobilized aptamer form effective ‘analyte–aptamer’ complexes that change the topography of the substrate surface. Therefore, the LOD of this LC-based BPA aptamer sensor was concluded to be 600 pM. According to the POM results, the correlation between the grayscale value and the BPA concentration was evaluated so as to analyze this system in a quantitative manner. The grayscale value of the LC regions was obtained by POM using the Adobe Photoshop CS6 software. As shown in Fig. 4, the grayscale values improved with the increasing concentration of BPA from 20 pM to 200 μM. The grayscale values increased rapidly against the BPA concentration logarithm from 600 pM to 20 nM. There was a slight fluctuation in the value of grayscale from 200 nM to 200 μM. This aptamer sensor had a linear range of 20–600 pM and 600 pM-30 nM, with linear equations Grayscale = 0.0171CBPA + 5.0941
So as to illustrate the selectivity of the aptasensor relying on the specific combination between the DNA probe and its target, two structurally similar compounds, BPB and BPC, were applied as interfering substances on the substrate membrane immobilized with the BPA aptamer to obtain LC cells. The concentrations of BPB and BPC employed were 10 times higher than that of BPA (20 vs 200 nM). The POM images for BPB (Fig. 5B) and BPC (Fig. 5C) display a uniformly black appearance with few low-brightness spots, while the LC system treated with BPA shows a more colored appearance (Fig. 5A). A blank control containing only TBS buffer (pH 8.0) was also analyzed, affording a completely black image (Fig. 5D). From these results, it is clear that BPB and BPC are incapable of triggering a large change in the alignment of 5CB molecules from a homogenous to random state, demonstrating that the aptasensor based on LCs has great selectivity toward BPA as the target molecule. 3.5. Topographical analysis by AFM AFM was employed for the characterization and verification of the nanometer-scale topographical changes on the sensor surface before and after exposure to BPA. All images in Fig. S4 were taken at 1.0 Hz scan rate and 1.0 μm scan size, and the 3D images were captured using a 5 nm mode height scale. The surface topography of the original film (a bare silicon wafer) is presented in Fig. S4A with an RMS roughness of 0.979 nm, indicating a smooth surface. After that, the sensor film was modified with (APTES+GA)/DMOAP and the roughness value of the surface increased to 1.159 nm (Fig. S4B), indicating a slightly rough surface. As shown in Fig. S4C, the RMS roughness was 1.701 nm after incubation with the BPA aptamer. Then, the effect of exposure to BPA on the BPA aptamer was investigated. The value of RMS significantly rose to 2.640 nm (Fig. S4D), i.e., an increase of 0.939 nm compared to that of the surface incubated solely with the BPA aptamer, indicating a considerable change on the surface. The results suggest that BPA specifically modifies the BPA aptamer. We can see further results in Fig. S4E and F: the topography of the surface incubated with BPB or BPC at a concentration 10 times higher than that of BPA was similar to that of 1068
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Fig. 4. Relationship between the grayscale values and BPA concentration. The grayscale values were obtained as the average of three repeated measurements for every assay with BPA( ± SD). The standard deviation (SD) is a measure that is used to quantify the amount of variation or dispersion of a set of data values [46].
the surface modified solely with the BPA aptamer, resulting in no significant changes in the RMS roughness. The results support the assumption that an LC orientational transition is only induced when BPA is bound to the aptamer immobilized on the surface, resulting in large changes in the surface topography.
doped orange juice were measured. For this purpose, the prepared samples were dropped on the sensor substrate for testing. As can be seen in Fig. 6A, B, and C, the bright appearance is derived from the random alignment of 5CB molecules after modification by the real BPA samples.
3.6. Detection of BPA in real samples
4. Conclusion
In order to validate the practicality of the aptamer-based sensor for BPA detection, real samples including a CD disc, plastic bag, and BPA-
A label-free LC-based aptamer sensor to detect BPA via a surfaceimmobilized aptamer was fabricated to study the specific binding
Fig. 5. POM images of LC sensing cells after binding between BPA aptamer (1 μM) and different substances: (A) 20 nM BPA, (B) 50 mM TBS buffer (pH 8.0), (C) 200 nM BPB, and (D) 200 nM BPC in TBS buffer (pH 8.0). Scale bar: 100 μm. 1069
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Fig. 6. Optical images of the LC cells captured under polarization for real samples: (A) a CD disc, (B) a plastic bag and (C) BPA-doped orange juice. Scale bar: 100 μm.
between said BPA aptamer and its target. The formation of a ‘BPA–aptamer’ complex on the substrate disrupts the surface topography, as well as induces a transition of the LC orientation. These results can be easily observed by POM owing to the birefringent characteristics of the LCs. At the same time, this work has demonstrated that BPA can be easily, rapidly, sensitively, and specifically detected using such aptamer-based LC sensor compared to other analytical methods that require complicated processes and instrumentation. Moreover, the AFM analysis supported the hypothesis that a perpendicular-to-random orientational transition of the LC molecules is induced by the specific binding between the BPA aptamer and its target. After treatment with BPA, significant changes in the roughness of the aptamer-immobilized surface were observed. The optimal conditions for the alignment of liquid crystal molecules were an APTES/DMOAP ratio of 5:1; 1% GA to anchor the BPA aptamer, and a minimum concentration of BPA aptamer of 1 μM to ensure adequate sensitivity of the aptamer sensor. The modified biosensor presents a limit of detection of 600 pM. Therefore, the present LC-based aptamer sensor may also be suitable for the rapid detection of other small molecules due to its advantages of being labelfree with simple operation, low cost, and high response rate.
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Liquid crystal-based aptamer sensor for sensitive detection of bisphenol A Huihui Ren, Zongfu An, Chang-Hyun Jang
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Department of Chemistry, Gachon University, Seongnam-daero1342, Sujeong-gu, Seongnam-si, Gyeonggi-do 13120, Republic of Korea
ARTICLE INFO
ABSTRACT
Keywords: Aptamer sensor Liquid crystal 4-Cyano-4′-pentybiphenyl Bisphenol A
Bisphenol A (BPA) is an environmental poison that seriously affects the health of humans. Herein, a novel, labelfree, time-saving liquid crystal-based aptamer sensor for the detection of BPA was developed based on the specific binding of an aptamer and its target. In this study, the surface of the sensor was assembled with (3aminopropyl)triethoxysilane & dimethyloctadecyl[3-(trimethoxysilyl) propyl]ammonium chloride and the amino-aptamer was attached to this film via glutaradehyde links. Through a specific reaction, BPA reacts with the BPA aptamer forming an ‘aptamer–BPA’ complex that changes the conformation of the BPA aptamer. Through spatial size effects, the large size of the complex disturbs the nematic orientation of 4-cyano-4′-pentybiphenyl in the film on this surface from homeotropic to random, resulting in an image that changes from ‘dark’ to ‘bright’ under a polarized light microscope. The limit of detection of this system is 600 pM. Atomic force microscopy was also employed to further validate the results.
1. Introduction Bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl)proane, C15H16O2) is an endocrine disrupting chemical bearing two phenol functional groups (Fig. 1A) that is considered an environmental hormone substance [1]. BPA has always drawn great concern because it is one of the main raw and processed materials in the manufacture of epoxy resins and polycarbonate (PC) [2]. It has also other applications such as in baby supplies, food-grade packaging materials, and pesticides [3,4]. It has been reported that BPA is released into the environment mainly through sewage effluents by leaching from the epoxy resin and commercial PC products during the corresponding industrial processes [5,6]. More importantly, many studies have shown that BPA has great impact on the human body related to damage of the reproductive function and other diseases [7,8], such as miscarriages, precocious puberty, prostate growth, cancer [9], being introduced in the human body via the food chain and enriched by bioaccumulation [10]. In addition, its excretion is difficult because BPA is not easily degraded [11]. Although the level of BPA in the environment is extremely low, it still causes blockage of the neural system, malformation and canceration of organs, functional disorders of the endocrine-immune system, and interference with the enzymatic system [12]. According to the public Health Service Food and Drug Administration (FDA), the tolerable daily intake value (TDI) is 5 mg/kg bw/day (23.126 μM) [13]. Thus, the development of rapid, sensitive, and simple methods to determine BPA levels is urgent. Various analytical methods have been developed to detect BPA,
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which include: gas chromatography–mass spectrometry (GC–MS) [14], liquid chromatography-mass spectrometry (LC-MS) [15], high-performance liquid chromatography (HPLC) [16], Enzyme-Linked Immunosorbent Assay (ELISA) [17], electrochemical technologies [18], and molecular imprinting methods [19]. However, most of these approaches need long times, expensive and complex instrumentation, or a specific labeling agent, and are thus not suitable for the detection of BPA. Therefore, the development of low-cost, efficient, high-sensitivity, fast, and label-free methods remains a big challenge. We have developed a liquid crystal-based aptamer sensor. It has a limit of detection (LOD) of 600 pM, and a linear range from 20 to 600 pM and from 600 pM to 30 nM. In order to illustrated the sensor performance, the characteristics of LC-based aptamer sensor were compared to other reported methods in Table S1. Ye et al. developed an electrochemiluminescence aptamer sensor for BPA detection in milk and water. The device used an Au electrode, which achieved a linear range from 0.1 to 100 pM and an LOD of 76 fM with detection time of 7 h [20]. Yu et al. presented electrochemical aptamer sensor using triple-signaling strategy to detect BPA in real-water samples in approximately 3 h. This sensor had an LOD of 0.19 pM with a linear range from 1 to 100 pM [21]. Kazane et al. reported electrochemical aptamer sensor based on Poly (Pyrrole-Nitrilotriacetic Acid)-Aptamer Film to detect BPA with detection time of 30 min. The device had a linear range from 10 pM to 1 μM with an LOD of 10 pM [22]. Zhu et al. developed a fluorescence aptamer sensor for detecting BPA in actual water samples. The device used a surface of graphene oxide and detect BPA from 0.1 to
Corresponding author. E-mail address: [email protected] (C.-H. Jang).
https://doi.org/10.1016/j.microc.2019.02.019 Received 27 September 2018; Received in revised form 30 January 2019; Accepted 7 February 2019 Available online 08 February 2019 0026-265X/ © 2019 Published by Elsevier B.V.
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phenomenon was found to be reversible [32]. Later, Lehmann also observed this phenomenon and found that the mesophase (the turbid liquid) presented similar properties to those of a crystal [33]. Thus, a liquid that presents fluidity and optical anisotropy is called an LC. The principle of LC-based biosensors relies on the unparalleled conformation of the LC line phases and the elastic, optical, anchoring, and birefringent properties of the LC material [34]. LC material has the property of birefringence that could be observed by polarizing microscope. There are two polarizing plates perpendicular to each other (the lower polarizer is called a polarizer and the upper polarizer is regarded as an analyzer). When natural light passes through the polarizer, it becomes linearly polarized light. As showed in Fig. 1B, when LCs are vertically aligned and the linearly polarized light passes through the LCs, the vibration direction of polarized light and the analyzer is perpendicular to each other. Because the vibration direction of the polarized light cannot be changed, the observed field is black. If the LCs are planar, there is a great change in the vibration direction of polarized light under the influence of LCs. At this time, part of the polarized light can go through the analyzer, so the brightness images could be obtained. Incorporation of receptors and ligands on the surface of the biosensor can disrupt the alignment of LC molecules [35]. This deviation can then be processed optically, and the arrangement of LCs can be correlated with the identity and/or concentration of the analyte. Most of these LC-based biosensors depend on the binding of biological macromolecules that change the alignment of the LCs on the surface. However, to date, only a few LC-based sensors have been proposed that can be used to detect small organic molecules [36]. This study reports a highly sensitive method to detect BPA using an LC-based aptamer sensor. Biosensors with biomolecules as sensing elements have become the most promising method for BPA detection because of their high response rate, high specificity, and simple operation [37]. Recently, antibodies have been extensively applied to discriminate biological elements; however, due to their lengthy preparation, and great instability, their application in biosensors remains limited [38]. Compared to traditional approaches based on antibodies, new recognition elements for aptamers [39], such as single-stranded DNA or RNA (ssDNA or ssRNA, respectively), can be screened from large mononucleotide databases through the Systemic Evolution of Ligands by Exponential Enrichment (SELEX) technique [40]. In addition, aptamers can selectively bind proteins [41], small organic molecules [42], antibodies [43], and even cells [44]. Recently, more and more recognition elements have led to the fabrication of biosensors, a field where aptamers have been gaining significance. In addition to highaffinity binding and high-specificity recognition characteristics, similar to those of antibodies, aptamers also exhibit superior properties such as low molecular weight, wide range of target molecules, easy repetitive synthesis and labeling, stability during storage, and susceptibility toward denaturation [45].
Fig. 1. (A) Chemical structure of BPA; (B) Schematic illustration of optical switching behaviors of 5CB.
10 ng/mL with an LOD of 0.05 ng/mL [23]. Chen et al. demonstrated a fluorescence aptamer sensor fabricated by construction of the DNA Y junction. The device could detect BPA concentration as low as 5 fM with detection time of 90 min [24]. Mirzajani et al. designed a PCBbased capacitive aptamer sensor for label-free analysis of BPA in canned foods with detection time of 20 s. The sensor could detect BPA from 1 fM to 10 pM with an LOD of 152.93 aM [25]. When compare to other techniques, our LC-based sensing approach possesses a range of characteristics that make it worthy of exploration. These characteristics include high speed, simple operation, low cost, and high sensitivity. Generation of optical outputs can be easily detected and quantified using visible light. It does not require laborious labeling, fluorescent or redox tags, or the need for complex instrumentation or highly skilled personnel. As for detection time, although the reaction between aptamer and BPA takes 1 h, amplification (reorientation of LC) can occur within a second. The amplification step does not need to be carefully timed, as in case of ELISA, thus simplifying the design of multiplexed analyses. Liquid crystals (LCs) are substances between the crystalline solid state and isotropic liquid state [26] that exhibit optical anisotropy and long-range orientation [27], and that can also be used to magnify and convert chemical signals into electrical signals [28]. These properties, coupled with their rapid response to external stimuli, render LCs very suitable as “sensing elements” [29]. The phenomenon of liquid crystallinity was discovered by Friedrich Reinitzer, a famous botanist, while studying cholesteryl benzoate (C6H5CO2C27H48, CB) [30] in the 1980s [31]. At that time, Reinitzer found that there were two distinct melting points in cholesteryl benzoate. At a temperature of 145.5 °C, the compound melts into a turbid liquid state but, when heated to 178.5 °C, the cloudy liquid changes into a transparent, isotropic liquid; this
2. Materials and methods 2.1. Materials (3-Aminopropyl)triethoxysilane (APTES), dimethyloctadecyl[3-(trimethoxysilyl) propyl]ammonium chloride (DMOAP), bisphenol A, bisphenol B (BPB), and bisphenol C (BPC) were purchased from SigmaAldrich (St. Louis, MI, USA). Hydrogen peroxide (30% w/v), methanol, ethanol (anhydrous), sulfuric acid, phosphate-buffered saline (PBS), Tris-buffered saline (TBS), and glutaradehyde (GA) were purchased from Daejung Chemicvals & Metals Co. Ltd., South Korea. The aptamer used in the experiments (5′/5AmMC6/CCG CCG TTG GTG TGG TGG GCC TAG GGC CGG CGG CGC ACA GCT GTT ATA GAC GTC TCC AGC3′) was synthesized by Integrated DNA Technologies. Nematic LCs of 4cyano-4′-pentylbiphenyl (5CB) were obtained from Tokyo Chemical Industry Co., Ltd. (Japan). Micro slide glasses were purchased from Fisher Scientific (Pittsburgh, PA). All aqueous solutions were prepared 1065
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with deionized (DI) water (18.2 MΩ cm−1) using a Milli-Q water purification system (Millipore; Bedford, MA).
about 1.5 cm × 1 cm. A DMOAP-coated glass slide was used as the top surface, with an APTES/DMOAP-coated glass slide as the bottom surface placed face to face. The two surfaces were spaced by two thin polyester films (Mylar, 19 μm). The reaction proceeded on the functionalized APTES/DMOAP-coated surface, where 5CB was introduced in the cells under heating (at approximately 60 °C). Subsequently, the optical cells were slowly cooled and then polarized light microscopy was used to analyze them.
2.2. Cleaning of substances The micro slide glasses were immersed in a newly prepared piranha solution (v(H2SO4)/v(H2O2) = 7:3) for 30 min at 85 °C to remove all organic contaminants under nitrogen atmosphere (0.2 MPa). After that, they were rinsed three times with DI water, ethanol, and methanol, dried under nitrogen flow, and stored in an oven at 110 °C overnight prior to being decorated with APTES/DMOAP.
2.8. Atomic force microscope (AFM) measurements The root mean square roughness (RMS) and morphology of the modified mixed APTES/DMOAP self-assembled single layer were characterized via AFM (NanoScope IIIa, Veeco Metrology, Santa Barbara, CA). The AFM images were captured at a scan rate of 1.0 Hz and scanning area of 1 μm × 1 μm.
2.3. DMOAP decoration The washed micro slide glasses were immersed in an aqueous solution containing 0.4% (v/v) DMOAP for 30 min at room temperature, and then rinsed with large amounts of DI water. The DMOAP-decorated slide glasses were dried under a stream of nitrogen gas and stored in an oven for 1 h at 110 °C.
2.9. Optical image acquisition by polarized light microscopy (POM) All images of the LC optical response were taken using a polarized light microscope (Eclipse LV100 POL; Nikon; Tokyo, Japan) equipped with a digital camera (DS-2Mv; Nikon; Tokyo, Japan). All the optical images were obtained using a 4× objective lens between the crossed polarizer and the analyzer at a resolution of 1600 × 1200 pixels, gain of 1.00×, and shutter speed of 1/10 s.
2.4. APTES/DMOAP decoration The cleaned glass slides were immersed in an ethanol solution containing 1% (v/v) DMOAP and 5% (v/v) APTES for 1 h at 80 °C, and then successively rinsed with a large excess of ethanol and DI water to remove any physically absorbed APTES/DMOAP. Next, the slides were dried under a stream of N2 and stored in an oven for 1 h at 110 °C. Finally, the slides were immersed in a 1% (v/v) GA aqueous solution for 30 min, rinsed with DI water, and dried under nitrogen flow.
3. Results and discussion 3.1. BPA detection method
2.5. BPA-aptamer immobilization
The detection mechanism of the present LC sensor is shown in Fig. 2, based on the high specificity of the aptamer and its target molecules. The aptamer configuration was labeled with an amino group (eNH2) at the 3′-end of the chain, which was used to form a stable amide bond with the aldehyde group of GA, so that the aptamer could be fixed on the substrate. The sensing film was constructed with two reagents (APTES and DMOAP) to provide a long chain favoring the vertical alignment of LC molecules and an active group for biomolecule immobilization. Upon coating with GA (Fig. 2B), the amino-aptamer was fixed on the APTES/ DMOAP-modified glass slides (Fig. 2C). Due to the specificity of the aptamer and its target molecule (BPA), BPA can be effectively captured on the aptamer-based surface. In this context, the BPA aptamer serves as an identification element: when it reacts with BPA, its conformation changes and an ‘aptamer–BPA’ complex is formed (Fig. 2D). The large size of the complex greatly increases the density at the bottom film, remarkably disturbing the alignment of LCs so that 5CB molecules undergo an orientation transition from homeotropic to random under these conditions (Fig. 2F). Owing to the birefringence effect of the LC molecules, changes in the brightness of the image are observed by POM and rapid detection of BPA is realized. In the absence of BPA molecules, the conformation of the aptamer remains in the original state (Fig. 2E). In this case, the aptamer as an interface is insufficient to trigger a random orientation profile and thus the optical response observed by POM reveals a uniform black image.
The BPA aptamer was immobilized on the glass slides using a GA cross-linking method. First, the BPA aptamer was dissolved in 10 mM PBS (pH = 7.4, 2.7 mM KCl, 137 mM NaCl) and stored at 4 °C for use. After that, the GA-activated glass slides were immersed in the prepared aptamer solution at room temperature for 1 h, then rinsed successively with PBS buffer and DI water, dried under a stream of N2, and finally stored in an oven for 1 h at 110 °C. 2.6. Specific binding between the BPA aptamer and BPA The BPA aptamer-bound glass slides were treated with 80 mM glycine in aqueous solution for 1 h in order to block the unreacted aldehyde binding sites on the glass slides. Afterwards, BPA solutions at different concentrations were dropped on the BPA aptamer-immobilized glass slides at room temperature for 1 h, then rinsed with 50 mM TBS buffer (pH = 8.0) and DI water to remove any physically bound molecules, and finally dried with N2 gas to produce the final platform. In order to verify the practicality of the developed sensor, real samples containing BPA (plastic products such as a CD disc and plastic bag) were tested. In short, 1.0 g of each plastic product was placed in 15 mL DI water and processed by ultrasonication for 1 h at 90 °C; after that, the samples were stirred at 90 °C for 48 h and finally filtered. The final samples were stored in a refrigerator at 4 °C. Orange juice doped with BPA was also analyzed. The orange juice sample was centrifuge at 3000g for 30 min to remove any particle, and then diluted with 10 mM PBS (pH 7.4) at the ratio of 1:1. We added BPA into the sample to obtain a 20 μM solution of BPA. BPA was then measured by the method described above. Each experiment was repeated three times.
3.2. Optimization of fundamental conditions The orientation of LCs on the DMOAP-modified surface can be influenced by APTES. We therefore investigated the effect of the self-assembly of APTES/DMOAP on the orientational response upon incubation in APTES/DMOAP mixtures at different ratios. As shown in Fig. S1, the darkness of the optical LC image decreased with the gradual increase of the APTES/DMOAP volume ratio until a bright image was obtained. When the proportion of APTES/DMOAP was 1:1, a completely dark stabilized background was observed, corresponding to a
2.7. Preparation of sandwich LC optical cells The optical cells were constructed with two different solid surfaces and the LCs sandwiched in-between. The size of the optical cells was 1066
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Fig. 2. Diagram of the LC sensing device for BPA detection: (A) clean glass slide, (B) immobilization of (APTES/GA) DMOAP for 30 min at room temperature on the glass, (C) immobilization of the BPA aptamer for 1 h at room temperature, (D) incubation with BPA for 1 h at room temperature, (E) vertical alignment of 5CB in the optical cell in the absence of BPA, and (F) random orientation of 5CB after treatment with BPA. POM photographs are also shown.
perpendicular orientation of the LC molecules (Fig. S1A). The results demonstrated that the presence of small amounts of APTES does not perturb the perpendicular alignment of LCs in the film. In Fig. S1B, a dark texture with some bright spots is observed for the APTES/DMOAP solution at 5:1 ratio. In addition, compared to Fig. S1A and B, many bright spots are observed at an APTES/DMOAP volume ratio of 10:1 (Fig. S1C). At a higher ratio of APTES/DMOAP (20:1), the number of bright spots in the POM image increases, almost covering the whole image (Fig. S1D). In order to obtain enough signal-to-background contrast and good self-assembly efficiency for BPA-aptamer anchoring, an APTES/DMOAP proportion of 5:1 was finally chosen to immobilize GA. The signal background is also influenced by the amount of GA activating the APTES/DMOAP self-assembled film. Optimization experiments were conducted at various concentrations of GA and the results are shown in Fig. S2. It was found that, at a concentration of GA (v/v) of 0.5% and 1% (Fig. S2A and B), the optical appearance of LC cells showed a uniform black image. In contrast, when the concentration of GA (v/v) reached 4% (Fig. S2C), the liquid crystal arrangement was disturbed and a bright texture appeared in the POM images. In order to ensure a sufficient number of aldehyde groups for immobilization of the BPA aptamer and weak background signal interference, 1% GA was employed in subsequent experiments. It is well known that the orientational profile of 5CB molecules can
also be influenced by the surface of aptamers coated on glass slides. In order to improve the signal-to-background contrast and obtain a zerointerference background, experiments were conducted to optimize the concentration of BPA aptamer. As seen in Fig. S3, the extent of colored areas in the optical image decreased with the concentration of BPA aptamer. In Fig. S3A and B, at low aptamer concentrations (100 nM and 1 μM), uniformly black optical appearance was observed, suggesting that such loadings of BPA aptamer molecules are unable to influence the arrangement of LC molecules, and that higher concentrations are needed to ensure that enough aptamer molecules are present to obtain adequate BPA detection. When the concentration of aptamer was increased to 5 μM and 10 μM, multiple bright colored areas were observed (Fig. S3C and D). A large number of BPA aptamers were immobilized on the LC-based sensor, which had a great influence on the alignment of the 5CB molecules, causing large changes in the intensity and brightness of the optical image. Upon further increasing the concentration of aptamer, where the LC random arrangement was maintained, a greatly colored texture was observed in the optical image. Therefore, a concentration of BPA aptamer of 1 μM was chosen to provide adequate sensitivity of the subsequent self-assembly. In summary, the optimal conditions for the alignment of liquid crystal molecules were determined to be an APTES/DMOAP ratio of 5:1; 1% GA to bind the BPA aptamer, and a minimum concentration of 1067
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Fig. 3. POM images of LC sensing cells sandwiched between an upper surface immobilized 0.4% DMOAP and a lower surface exposed to various concentration of BPA: (A) 20 pM; (B) 200 pM; (C) 600 pM; (D) 2 nM; (E) 20 nM; (F) 200 nM; (G) 2 μM and (H) 20 μM in TBS. Scale bar: 100 μm.
(R2 = 0.9807) and Grayscale = 54.377 logCBPA-139.89 (R2 = 0.9952) respectively. CBPA means the concentration of BPA. Based on the obtained image, a gradation analysis of the images can be performed and therefore, the quantitative analysis of BPA can be realized within a certain concentration range.
BPA aptamer of 1 μM to ensure the sensitivity of the aptamer sensor through the rearrangement of the liquid crystal alignment from homeotropic to random. 3.3. Limit of detection
3.4. Selectivity
In view of the fact that the anisotropic surface topography of the substrate is significantly affected by BPA, a study of the specific interaction between BPA and the BPA aptamer by cross-polarized optical imaging was performed. The configuration of the BPA aptamer, as the specific recognition element of the LC-based sensor, changes upon binding BPA. According to these results, we hypothesized that the alignment of 5CB molecules would be perturbed by the formation of a ‘BPA–aptamer’ complex via hydrogen bonding and ion pairing. Hence, a series of concentrations of BPA were tested under this assumption and optical images were obtained with a polarized light microscope. As shown in Fig. 3, the intensity and region of defects gradually increased to yield a completely bright texture with the increasing BPA concentration. A fully black optical appearance was obtained at BPA concentrations of 20 and 200 pM (Fig. 3A and B), showing that such BPA loadings are unable to perturb the perpendicular orientation of the surface. In Fig. 3C, at a BPA concentration of 600 pM, some obvious bright spots in the optical images were detected compared to Fig. 3A and B. In contrast, the optical response of LC cells displayed apparent brightness at BPA concentrations of 2 nM, 20 nM, 200 nM, 2 μM, and 20 μM (Fig. 3D, E, F, G and H respectively). This is due to high concentrations of BPA being able to disrupt the alignment of 5CB molecules, making the LC optical image bright. These results are consistent with our assumption that specific binding of BPA molecules and the surface-immobilized aptamer form effective ‘analyte–aptamer’ complexes that change the topography of the substrate surface. Therefore, the LOD of this LC-based BPA aptamer sensor was concluded to be 600 pM. According to the POM results, the correlation between the grayscale value and the BPA concentration was evaluated so as to analyze this system in a quantitative manner. The grayscale value of the LC regions was obtained by POM using the Adobe Photoshop CS6 software. As shown in Fig. 4, the grayscale values improved with the increasing concentration of BPA from 20 pM to 200 μM. The grayscale values increased rapidly against the BPA concentration logarithm from 600 pM to 20 nM. There was a slight fluctuation in the value of grayscale from 200 nM to 200 μM. This aptamer sensor had a linear range of 20–600 pM and 600 pM-30 nM, with linear equations Grayscale = 0.0171CBPA + 5.0941
So as to illustrate the selectivity of the aptasensor relying on the specific combination between the DNA probe and its target, two structurally similar compounds, BPB and BPC, were applied as interfering substances on the substrate membrane immobilized with the BPA aptamer to obtain LC cells. The concentrations of BPB and BPC employed were 10 times higher than that of BPA (20 vs 200 nM). The POM images for BPB (Fig. 5B) and BPC (Fig. 5C) display a uniformly black appearance with few low-brightness spots, while the LC system treated with BPA shows a more colored appearance (Fig. 5A). A blank control containing only TBS buffer (pH 8.0) was also analyzed, affording a completely black image (Fig. 5D). From these results, it is clear that BPB and BPC are incapable of triggering a large change in the alignment of 5CB molecules from a homogenous to random state, demonstrating that the aptasensor based on LCs has great selectivity toward BPA as the target molecule. 3.5. Topographical analysis by AFM AFM was employed for the characterization and verification of the nanometer-scale topographical changes on the sensor surface before and after exposure to BPA. All images in Fig. S4 were taken at 1.0 Hz scan rate and 1.0 μm scan size, and the 3D images were captured using a 5 nm mode height scale. The surface topography of the original film (a bare silicon wafer) is presented in Fig. S4A with an RMS roughness of 0.979 nm, indicating a smooth surface. After that, the sensor film was modified with (APTES+GA)/DMOAP and the roughness value of the surface increased to 1.159 nm (Fig. S4B), indicating a slightly rough surface. As shown in Fig. S4C, the RMS roughness was 1.701 nm after incubation with the BPA aptamer. Then, the effect of exposure to BPA on the BPA aptamer was investigated. The value of RMS significantly rose to 2.640 nm (Fig. S4D), i.e., an increase of 0.939 nm compared to that of the surface incubated solely with the BPA aptamer, indicating a considerable change on the surface. The results suggest that BPA specifically modifies the BPA aptamer. We can see further results in Fig. S4E and F: the topography of the surface incubated with BPB or BPC at a concentration 10 times higher than that of BPA was similar to that of 1068
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Fig. 4. Relationship between the grayscale values and BPA concentration. The grayscale values were obtained as the average of three repeated measurements for every assay with BPA( ± SD). The standard deviation (SD) is a measure that is used to quantify the amount of variation or dispersion of a set of data values [46].
the surface modified solely with the BPA aptamer, resulting in no significant changes in the RMS roughness. The results support the assumption that an LC orientational transition is only induced when BPA is bound to the aptamer immobilized on the surface, resulting in large changes in the surface topography.
doped orange juice were measured. For this purpose, the prepared samples were dropped on the sensor substrate for testing. As can be seen in Fig. 6A, B, and C, the bright appearance is derived from the random alignment of 5CB molecules after modification by the real BPA samples.
3.6. Detection of BPA in real samples
4. Conclusion
In order to validate the practicality of the aptamer-based sensor for BPA detection, real samples including a CD disc, plastic bag, and BPA-
A label-free LC-based aptamer sensor to detect BPA via a surfaceimmobilized aptamer was fabricated to study the specific binding
Fig. 5. POM images of LC sensing cells after binding between BPA aptamer (1 μM) and different substances: (A) 20 nM BPA, (B) 50 mM TBS buffer (pH 8.0), (C) 200 nM BPB, and (D) 200 nM BPC in TBS buffer (pH 8.0). Scale bar: 100 μm. 1069
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Fig. 6. Optical images of the LC cells captured under polarization for real samples: (A) a CD disc, (B) a plastic bag and (C) BPA-doped orange juice. Scale bar: 100 μm.
between said BPA aptamer and its target. The formation of a ‘BPA–aptamer’ complex on the substrate disrupts the surface topography, as well as induces a transition of the LC orientation. These results can be easily observed by POM owing to the birefringent characteristics of the LCs. At the same time, this work has demonstrated that BPA can be easily, rapidly, sensitively, and specifically detected using such aptamer-based LC sensor compared to other analytical methods that require complicated processes and instrumentation. Moreover, the AFM analysis supported the hypothesis that a perpendicular-to-random orientational transition of the LC molecules is induced by the specific binding between the BPA aptamer and its target. After treatment with BPA, significant changes in the roughness of the aptamer-immobilized surface were observed. The optimal conditions for the alignment of liquid crystal molecules were an APTES/DMOAP ratio of 5:1; 1% GA to anchor the BPA aptamer, and a minimum concentration of BPA aptamer of 1 μM to ensure adequate sensitivity of the aptamer sensor. The modified biosensor presents a limit of detection of 600 pM. Therefore, the present LC-based aptamer sensor may also be suitable for the rapid detection of other small molecules due to its advantages of being labelfree with simple operation, low cost, and high response rate.
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