1. Introduction

The quality deterioration of heavy metal ions in natural environment has become a major global issue. With developed of industry and technology, more and more metal ions have been continuously released [1,2]. Excess heavy metal ions can be a hazard in drinking water directly the aquatic ecosystem and human health [3]. Lead ions (Pb²⁺) are one of the major environmental pollutants that are dangerous for human health including memory loss, muscle paralysis, mental troubles, and cardiovascular dysfunction [4,5]. Therefore, sensitive and quantitative detection of lead ions is quite important. Several methods have been developed for the detection of lead ions, such as inductively coupled plasma atomic emission spectroscopy (ICP-AES) [6], atomic absorption spectroscopy (AAS) [7,8], surface plasmon resonance (SPR) [9], colorimetric [10,11], biochemical [12] and electrochemical techniques [13,14]. However, these analytical methods require highly expensive instruments, cumbersome operating conditions and time-consuming pretreatments. Therefore, it is highly desirable to develop novel method for Pb²⁺ real-time detection. As a naked-eye optical sensor, liquid crystals (LCs) based optical sensor has attracted significant attention due to its fast, low-cost, and simplicity [15]. The naked-eye LC optical sensor for Pb²⁺ detection is a promising technique in clinical toxicology, environmental monitoring and industry process monitoring applications.

In recent years, liquid crystals have attracted a lot of interests in chemical [16], material [17] and biological fields [18] due to their unique optical and optoelectronic properties. The liquid crystal is extraordinary sensitive to physical and chemical properties of a bounding interface due to the transduction ability of molecular events at interface [19–21]. LC based sensor has been widely used in various chemical analysis and biosensors. Abbott et al. reported the measurement of chemical exposure based on recognition-driven anchoring transitions in LC, and then they demonstrated the gas sensors using metal salt immobilized liquid crystals [22]. Several research works have been demonstrated to utilize nematic liquid crystals such as 5CB for detection of bacteria [23,24], viruses [25], cancer cells and protein [26], polymer chemicals [27] and heavy metal ions [28].

Herein, we developed a liquid crystal optical sensor for detection of Pb²⁺ based on DNA degradation. The mechanism of detection is due to the disturbance of ordered liquid crystals configuration induced by adding Pb²⁺. The proposed LC optical sensor possesses a wide detection range with low detection limit, and a high selectivity, which leads to a quickly, simply and low-cost quantitative analysis of Pb²⁺.

DNAzyme (catalytic strand), as the main member of functional nucleic acids, is reported to recognize metal ions, due to the capability of either binding to a target molecule or performing catalytic reactions [29]. In 2003 Brown reported a DNAzyme that was used for binding Pb²⁺ and result in catalytic hydrolysis. Several types of DNAzyme based Pb²⁺ sensor have been reported. DNAzyme has been for binding Pb²⁺ and resulting in catalytic hydrolysis of the nucleic acids [30]. A colorimetric sensor by utilizing Pb²⁺ induced allosteric G-quadruplex DNAzyme for detection the Pb²⁺ was developed [31]. Both electrochemical and photoelectrochemical DNAzyme sensor for detection of Pb²⁺ have been demonstrated [32]. “Turn on” fluorescence sensor based on graphene quantum dots and gold nanoparticles has also been studied [33]. In this study, DNAzyme and its combined strand were used for LC based optical Pb²⁺ sensor. Figure 1(a) depicts the fabrication process of the Pb²⁺ sensor based on liquid crystals. Firstly, a glass slide was modified with N, Ndimethyl-N-octadecyl (3-aminopropyl) trimethoxysilyl chloride (DMOAP) and 3-aminopropyl-trimethoxysilane (APTES). The DMOAP was used to align liquid crystals (5CB) molecules perpendicularly to the surface of glass substrate through chemical bonds of molecules interactions [12], leading to a homeotropic configuration of liquid crystals on the glass slide. The APTES molecules were attached on the substrate surface of glass by covalent Si-O-Si bonds. It possessed plentiful of terminal amino groups, could catch specific anionic complementary DNA strands (the structural formula is shown in Fig. 1(b)) that consisted of catalytic strand (DNAzyme) and combined strand. Glutaraldehyde (GA) was subsequently added as the intermedia of APTES and DNA strand, where the amino groups in GA and APTES would cross linked with each other. Then a cationic surfactant dodecyl trimethyl ammonium bromide (DTAB) and nematic liquid crystal 5CB were added on the surface of glass slide. The DTAB (aqueous solution) molecules could bind to the DNA through electrostatic interaction [34], resulting in good dissolution of DNA in aqueous solution. It would form a self-assembly monolayer at the aqueous/LC interface and assist the alignment of LC molecules [35]. The liquid crystal 5CB molecules (as well as DNA strand) would perpendicularly align on the DMOAP coated glass substrate and form homeotropic configuration, leading to a dark optical image under polarizing optical microscope (POM). In appearance of Pb²⁺, the complementary DNA strand would disassemble by catalytically cleaving of DNA molecules due to catalytic activity of catalytic strand. The DNA molecules would be catalytically cleaved at the “rA” site [34], unwinding the complementary DNA strand with nucleotide fragment, as shown in Fig. 1(c). The cleaved DNA strands would disturb the homeotropic orientation of LC 5CB, leading to a bright optical image under POM.

 

Fig. 1. (a) Schematic illustration of Pb2+ detection based on liquid crystals. (b) Structure formula of complementary DNA strands. (c) Schematic of catalytically cleave of the complementary DNA molecules.

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2. Materials and methods

In our experiment, sodium citrate solution, phosphate buffer solution (10 mM PBS, contained with Na₂HPO₄ and NaHPO₄, pH 7.4), Pb(NO₃)_2, AgNO_3, KCl, HgCl, CuCl₂, MnCl_2, CaCl_2, ZnCl₂, CdCl_2, DMOAP, APTES, DTAB and GA were all obtained from Sigma-Aldrich (St. Louis, USA). Liquid crystal 5CB was bought from HCCH (Jiangsu, China). The combined strand and catalytic strand were purchased from Shenggong Bioengineering (Shanghai, China), and glass slides (Sail brand) were purchased from Dongsheng Glass (Taizhou, China). Optical images were obtained by polarization optical microscope (Ti 200, Nikon). The catalytic strand (5’-NH₂-CGATAACTCACTATrAGGAAGAGATG-3’), and combined strand (5’-CATCTCTTCTCCGAGCCGGTCGAAATAGTGAGT-3’) were purchased from Shenggong Bioengineering (Shanghai, China).

To fabricate the Pb²⁺ sensor, the glass slide was cleaned by sufficient piranha solution (1:1 30% H₂O₂ in H₂O:H₂SO₄; caution: piranha solution is exothermic and strongly reacts with organics) at 110°C for 30 min and then copiously rinsed with deionized water. After drying by nitrogen (N₂), the sample was immersed in ethanol solution that contained 0.1% (v/v) DMOAP and APTES for 30 min. Then, 0.5% GA was coated on the substrate and dried under N₂. Afterwards, the sample was immersed in a solution that contained 200 µM catalytic strand and 200 µM combined strands of DNA in 37°C for 1 h. Subsequently, the sample was rinsed with deionized water to clean unbonded DNA strands. Finally, transmission electron microscopy (TEM) grids were placed on the surface of glass slide and 1 µL of 5CB was dispensed onto each grid by dropping. Then, the 5CB-filled TEM grid was exposed in DTAB solution. All our experiments are carried on room temperature of 25°C. In our experiment, the thickness of LC is determined by the thickness of TEM grids, which is 10 µm.

3. Results and discussions

The concentration of DTAB plays an important role in alignment of liquid crystals with DNA strands in the initial state. Figure 2 shows the optical image of Pb²⁺ sensor sample under polarization optical microscopy (POM) at increase of DTAB from 0 M to 50 Mm. It can be seen that, when there was no DTAB (0 M), the optical image of sample was bright. The brightness decreased with the increase of concentration of DTAB, indicating that more and more LC molecules had been perpendicularly anchored on the glass slide. When the concentration of DTAB reached to 10 mM, the optical image of sample turned to almost totally dark except the edge of grid. Further increase of the concentration of DTAB up to 50 mM wouldn’t significantly change the darkness of sample. It means that the alignment effect of DTAB saturates from 10 mM, where the cationic from DTAB and anionic from complementary DNA strand comes to anion-cation balance. In our experiment, the 10 mM of DTAB was chosen as the optimal concentration in following parts. In Fig. 2, the POM images of LC are measured in a stable status (same time gap is used for keeping measure condition fixed) in different concentration of DTAB. The stable condition is also applied in Fig. 3 for influence of DMOAP.

 

Fig. 2. Optical appearances of Pb²⁺ sensor sample under polarizing optical microscope with (a) 0 M, (b)100 µM, (c)250 µM, (d)500 µM, (e)750 µM, (f)1 mM, (g) 10 mM, and (h) 50 mM concentration of DTAB.

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Fig. 3. POM image of sensor in case of (a) without DMOAP and (b) with DMOAP.

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To study the influence of DMOAP for anchoring of 5CB molecules, two glass slides were used in comparison experiment, where one glass slide was modified with APTES/GA and another was modified with DMOAP/APTES/GA. Figure 3(a)–3(b) show the POM image of sample cell when the complementary DNA strand and 5CB was added in the LC cell in case of without DMOAP and with DMOAP, which corresponds to a bright and dark image, respectively. It is indicated that the DMOAP plays a great role of anchoring 5CB molecules in the strategy for forming the homeotropic configuration of LC molecular. In addition, it is worth noticing that the concentration (from 0 to 500 µM) of the complementary DNA strand has no significant effect on the anchoring of 5CB in our experiment.

To evaluate the analytical performance for quantitative analysis of Pb²⁺, the effect of Pb²⁺ concentration on brightness of sensor POM image was investigated. Different concentration of Pb²⁺ (0-500 µM) solution was added into the optical sensor based on liquid crystal. Before the Pb²⁺ was added (Fig. 4(a)), the optical image was relatively dark, indicating a good homeotropic configuration of LC molecules. However, after the Pb²⁺ was added into the sensor, the complementary DNA strand would disassemble by catalytically cleaving of DNA molecules. The cleaved DNA strands would disturb the homeotropic orientation of LC 5CB, leading to a bright optical image under POM. The amount of cleave DNA strands depends on the concentration of Pb²⁺ added. With the increase of Pb²⁺, more and more DNA strands were cleaved, resulting in a brighter optical image under POM. Therefore, the brightness could be used as the indicator for the added concentration of Pb²⁺. Figure 4(b)–4(h) shows the optical image of sensor under POM when concentration of Pb²⁺ was 50 nM, 150 nM, 500 nM, 1 µM, 50 µM, 250 µM and 500 µM, respectively. The relationship between optical image brightness and the logarithm of concentration of Pb²⁺ is plotted in Fig. 4(i). The initial average gray value of grid is denoted by G₀, which is corresponding to the case of no Pb²⁺ presence as shown in Fig. 4(a). With the increase of Pb²⁺, the image becomes brighter and the value of G decreases accordingly, therefore, the relative intensity increment with the expression (G₀-G)/G₀ demonstrates a dynamic range of Pb²⁺. It is noticed that, as the surface tension between the grid edges and LC molecules, some LC molecules will be adsorbed on the TEM grid edges and gather together form a random arrangement, leading to inevitable light leakages even in homeotropic LC configuration. It can be expressed by y = 0.1893x-0.2998, where the Pb²⁺ sensor has a broad dynamic range of 50 nM- 500 µM. In this system, the dynamic range of the Pb²⁺ is affected by concentration of DNA strand. Thus the the increase of the concentration of DNA strand might lead to wider dynamic range. The detection limit was obtained about 36.8 nM according to the 3σ criteria, where σ is the standard deviation based on calculating three parallel blank signals and M is the slope between signal and sample concentration. A similar work is reported by Mazumdar et al., where they useed the 8-17 DNA strand and AuNPs to construct an easy-to-use dipstick test for Pb²⁺ and achieved a detection limit of 500 nM [36]. Xiao et al. developed an electrochemical biosensor based on DNAzyme to detection Pb²⁺ with a detection limit of 300 nM [37]. Wang et al. demonstrated a polydiacetylene-based sensor for Pb²⁺ with a limit of 1 µM [38].

 

Fig. 4. The optical image under POM for Pb²⁺ sensor when the concentration of Pb²⁺ is (a) 0 nM, (b) 50 nM, (c) 150 nM, (d) 500 nM, (e) 1 µM, (f) 50 µM, (g) 250 µM, and (h) 500 µM, respectively. (i) The relationship between optical image brightness (G₀-G)/G₀ versus a series of Pb²⁺ concentration of from 50 nM to 500 µM.

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Another important performance parameter of sensor in practical application is the selectivity. Figure 5 demonstrates the selectivity of Pb²⁺ sensor at presence of 100 µM Ca²⁺, Mg²⁺, Al³⁺, Cd²⁺, K⁺, Mn²⁺, Cu²⁺, Zn²⁺, Fe³⁺, Hg²⁺, Ag⁺ and 1 µM Pb²⁺. It can be seen that the optical image brightness was sharply increased that was induced by addition of 1 µM Pb²⁺, while no significant change had been observed after the addition of 100 µM other metal ions. The restoration efficiency of Pb²⁺ was about 4 times to other metal ions. This optical sensor is helpful to distinguish Hg²⁺ and Pb²⁺ as it is not easy to identify them. The system shows negligible response of Ca²⁺, Mg²⁺, Al³⁺, Cd²⁺, K⁺, Mn²⁺, Cu²⁺, Zn²⁺, Fe³⁺, Hg²⁺, and Ag⁺ under the similar condition. The results indicates that other metal ions aforementioned couldn’t cleave the DNA strands and disturb the LC homeotropic configuration, therefore there is no much change of optical image. The result shows that the sensor possesses a remarkable high sensitivity to Pb²⁺. This is the first time to achieve high sensitive and selective optical sensor for Pb²⁺ detection based on cleave of DNA strands induced change of liquid crystals configuration.

 

Fig. 5. Selectivity of the sensor in presence of Ca²⁺, Mg²⁺, Al³⁺, Cd²⁺, K⁺, Mn²⁺, Cu²⁺, Zn²⁺, Fe³⁺, Hg²⁺, Ag⁺ (100 µM) and Pb²⁺ (1 µM).

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4. Conclusions

In this work, a novel liquid crystal optical sensor for detection of Pb²⁺ based on DNAzyme and its combined strand has been demonstrated. The ordered and disordered configuration of liquid crystals, induced by complementary DNA strand and catalytically cleaved DNA in presence of lead ion separately, leads to dark and bright optical image under POM. The proposed naked-eye optical sensor possesses an extremely broad detection range of Pb²⁺ from 50 nM to 500 µM, with a low detection limit about 36.8 nM. The sensor also demonstrates high selectivity of Pb²⁺ from many other metal ions. The proposal LC sensor is high selective and sensitive for Pb²⁺ detection, which provides a novel platform for other heavy metal, DNAs or antigen in biological and chemical fields by modifying sensing molecules.