In this study, a novel liquid crystal (LC) biosensor was developed for the highly sensitive and selective detection of Cd 2+ based on Cd 2+ inducing the bending of PS-oligo. This strategy makes use of the DNA conformational change to enhance the disruption of orientation of LC leading to an amplified optical signal. DNA containing-SH was bound on the glass slide of the LC cell modified with the DMOAP/APTES. The DMOAP can effectively induce the homeotropic alignment of LC. In the presence of Cd 2+, Cd 2+ can induce DNA to bend and become a 2 nm spherical structure, which can greatly disrupt the orientational arrangement of LC, resulting in the correspond changes of the optical image of LC cell birefringent under the polarizing microscope. When the Cd 2+ concentration is low to 0.1 nM, the optical signal of LC biosensor has an obvious change. But in the absence of Cd 2+, there is no orientational response of LC and the optical image under the polarizing microscope is still a uniform dark background. Thus, this LC sensing method has a sensitive and clear distinction between positive and negative results and offers a highly sensitive detection of Cd 2+ with a low detection limit down to 0.1 nM.
Cadmium (Cd) is a heavy metal environmental toxicant which is widely present in air, soil, water and food. It can be accumulated in the human body mainly in the lungs, liver, kidney, brain, heart and testis with a biological half-life of greater than 20 - 30 years [
The instrument analysis method is a common method for Cd2+ detection, including electrochemical stripping analysis, atomic absorption spectroscopy (AAS) [
In order to improve the selectivity, recently, the nucleic acid aptamer (or oligonucleotide fragments) widely concerned is an ideal molecular recognition element on account of its some advantages including the broader universality than antibodies, specificity and affinity comparable to antibody, large-scale and low-cost production, high extremely stability, used repeatedly, etc. [
How to improve the detection sensitivity of Cd2+ (lower detection limit) is another key problem that must be solved in biological sensor. In recent years, Wu [
Therefore, combining the DNA specificity and the advantages of LC biosensors, we exploit a high specificity and sensitivity LC biosensing technique using DNA as specific recognition for Cd2+ detection.
N, N-dimethyl-N-octadecyl-3-aminopropyltrimethoxysilyl chloride (DMOAP) and (3-aminopropyl) triethoxysilane (3-APTES) were purchased from Sigma-Aldrich. LC 4-cyano-4’-pentylbiphenyl (5CB) was obtained from Huajing Scientific and Technological Development Co., Ltd. (Hebei, China). Glutaraldehyde (GA) was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Glass sides were provided by Xinhua Laboratory Glassware Company (Haimen, China). All metal ions were purchased from Chemical Reagent Co. WELDECH (Shanghai China). Oligonucleotides were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian China). Coppers contained network were purchased from Beijing Zhongjingkeyi Technology Co., Ltd. (Beijing, China). All chemicals used in this work were analytical reagent. Ultrapure water was used throughout all experiments.
The sequence of DNA employed is as follows:
5'-G-(SH)-GGGGGGGGG-(CH2)6-NH2-3'.
The glass sides were firstly cut into size of 2 cm × 2 cm square, and then the glass slides were cleaned with freshly prepared piranha solution (70% H2SO4, 30% H2O2) at 80˚C for 1 h. The glass slides were then rinsed with ultrapure water and ethanol for 5 - 6 times, dried with nitrogen, and stored in a 110˚C vacuum oven for 3 h before use.
1) DMOAP-decoration. Cleaned glass slides were immersed in an aqueous solution containing 0.35% (v/v) DMOAP for 30 min and then rinsed with ultrapure water. The DMOAP-coated glass slides were dried with nitrogen and heated in a 110˚C vacuum oven for 1 h.
2) DMOAP/APTES-decoration. Cleaned glass slides were immersed in ethanol solution containing 0.3% (v/v) DMOAP and 3% (v/v) APTES at 80˚C for 2 h, rinsed with ethanol and ultrapure water, respectively, dried with nitrogen, and then stored at 110˚C for 1 h. Subsequently, the sides were immersed in a 0.3 mol/L GA solution for 30 min and rinsed with ultrapur water, dried with nitrogen.
All DNA employed in this work were first dissolved in a 10 mM HEPES buffer (pH 7.0) containing 0.03 M NaCl, 0.03 M trisodium citrate 200 μL 100 nM solution of capture probe was dropped to the DMOAP/APTES/ GA-decorated glass slides, then incubation at room temperature for 1h, rinsed with a solution of 0.3 M sodium chloride/0.03 M trisodium citrate (2× SSC, pH 7.0) containing 0.01% (v/v) SDS and ultrapure water.
The LC cells were fabricated by spacing a DMOAP-coated glass and a DMOAP/APTES-coated glass with two thin strips of Mylar (~23 μm), and then held together with small binder clips. The DMOAP-coated glass was used to induce the homeotropic alignment of liquid crystals, while the DMOAP/APTES-coated glass was used to covalent attachment of amine-labeled capture probes. The cells were placed at 40˚C for 5 min. The LC material, 5CB, heated into its isotropic phase was spontaneously drawn into each LC cell by capillary action. The LC cells were then slowly cooled to ~28˚C.
100 μL mixture solution contains different concentrations of Cd2+ were dropped on the DNA decorated glass slides at room temperature for 1 h. The unbounded DNA probes were rinsed with 2× SSC containing 0.01% (v/v) SDS and abundant ultrapure water. The optical appearance was examined using a polarized light microscope (Shaihai Changfang Optical Instrument, China) in transmission mode under crossed polarizer. The images were captured by a digital camera mounted on the microscope.
In selectivity experiments, Cd2+ ions were simply displaced substitute or mixed by other metal ions under the same conditions (i.e., K+, Ca2+, Co2+, Al3+, Zn2+, Cr3+, Mn2+, Cu2+ and Pb2+).
The detection mechanism of this LC biosensing technique is shown in
surface on a pretreated plane slide was firstly obtained by self-assembling the DMOAP, APTES and GA film (
To improve the signal-to-background contrast, we studied the background signal affected by the self-assembling modification of DMOAP/APTES mixture with different volume ratio. As can be seen from
DNA concentrations can also affect the signal-to-background contrastl, which would affect the sensitivity of the sensor. So the concentration of DNA was optimized in this test. As can be seen from
When the DNA concentrations is higher than 100 nM (150 nM or 200 nM), the excessive amount of DNA immobilized on the substrate surface can greatly affect the orientation of the LC molecules, resulting in the bright spots and birefringent textures appeared in the optical images (
The solution containing different concentrations of Cd2+ was added onto the DNA-modified under glass slide, after being treated, the lower glass slide combined with the DMOAP-coated upper glass slide face to face and the liquid crystal cell was obtained. As shown in
To test selectivity of Cd2+ , 0.1 nM Cd2+ and 1 nM other metal ion, including K+, Ca2+, Co2+, Al3+, Zn2+, Cr3+, Mn2+, Cu2+ and Pb2+ were examined under the same conditions. As displayed in
signal response for other metal ions and their optical images display a uniform dark background, while the bright spots and birefringent textures in the optical images of Cd2+ are observed. This experimental result indicates that the LC biosensor based on DNA aptamer has a high selectivity for Cd2+ detection.
In summary, we have proposed a new LC biosensor to detect Cd2+ with high sensitivity and selectivity based on Cd2+ inducing DNA containing-SH to bend and become a 2 nm spherical structure. The sensitivity of this method for the detection of Cd2+ can achieve 0.1 nM. To our best knowledge, this is the first demonstration only using target DNA induced by Cd2+ to produce enhanced optical signal in the field of LC biosensors. In comparison with the previous standard methods, this proposed sensor is simple, low cost and does not require costly instrument. So this sensor is well suitable for on-site and real-time detection for Cd2+ and it will have great potential for practical applications.
This work was financially supported by the National Natural Science Foundation of China (No.30500125 and No.31201047).
ShixiongDeng,QifengJiang,TingtingZhang,XingliangXiong,PingChen, (2015) Liquid Crystal Biosensor Based on Cd2+ Inducing the Bending of PS-Oligo for the Detection of Cadmium. Health,07,986-993. doi: 10.4236/health.2015.78116