Study Of Liquid Crystal Biosensing Method For DNA And Small Molecular Substances Assay

Liquid crystal (LC) is a special state of matter intermediate between crystallinesolid and isotropic liquid，which possesses long-range orientational order and opticalanisotropy and can be used to amplify and transduce the surface binding events intooptical outputs that can be easily observed. These idiosyncrasies, combined with theirfast response to an external stimulus, make it well-suited as “sensing element”. Theprinciples of LC-based biosensors rely on the optical, anchoring, and birefringentproperties of the LC material. The alignment of LC molecules can be disrupted by theintroduction of the binding event of ligands and receptors at the biosensor surfaces.The resulting distortion can then be addressed by optical methods, and the changes inthe original alignment are correlated with either the identity of the target and itsconcentration. Compared to traditional analytical approaches, the LC-based sensorspermit label-free detection with high sensitivity and without the requirement ofcomplex instruments and even the need of electrical power, making them sufficientlysimple and well suited for the primary screening assay of analytes performed awayfrom central laboratories. Most of these LC-based detections rely on the biologicalmacromolecule binding events which can change the anchoring behaviors of LC on thesurface, but as yet, few LC-based sensors have been proposed for the detection ofheavy metals ions and other small molecules. In addition, in spite of the versatility andsimplicity of these LC biosensors based on direct biomolecular binding events, therelatively low sensitivity is a detriment and limits their application in bioassays of lowconcentration or trace analytes. Hence, it is an important domain that in seekingsignal-enhancement strategies for the LC-based biosensing techniques to circumventthe problem of detection sensitivity. Typically, the design of the LC-based biosensorincludes three key steps: modification of substrates, interaction with the targetmolecule and signal transduction of the molecular recognition event. Focused on thesebasic items in the development of high-performance LC-based biosensors, theextending application of LC-based biosensor to small molecule analysis and the designof novel strategies for amplifying sensing signals have been investigated in the presentdissertation and described as follows:In chapter2: Due to the large specific surface area, good biocompatibility andsurface characteristics of nucleic acids of high load, a novel signal enhanced liquid crystal biosensor based on using gold nanoparticles (AuNPs) has been developed forhighly sensitive DNA detection. Firstly, a chemically functionalized surface on a plainglass slide was obtained by self-assembling an (3-aminopropyl) trimethoxysilane (APS)/N, N-dimethyl-N-octadecyl-3-aminopropyltrimethoxysilyl chloride (DMOAP) film,which cannot only provide plentiful amino groups for coupling with DNA probes butalso orientate LC perpendicularly to provide a dark background. Then, the DNAimmobilization was performed by binding the capture DNA probe to the APS/DMOAPfilm through a glutaraldehyde (GA) cross-linker, followed by hybridization of thetarget DNA and reporter DNA-functionalized AuNPs. The AuNPs-mediated DNA cangreatly change the surface density of DNA strands and increase the disruption of theorientation profile in the thin LC layer, resulting in a significantly lower detectionlimit (0.1pM). This biosensor not only significantly decreases the detection limit, butalso ofers a simple detection process and shows a good selectivity to distinguishperfectly matched target DNA from two-base mismatched DNA.In chapter3: Using K-ras gene mutation in codon12(GGT to GTT) as a model,we have developed a new LC biosensor for target gene containing SNP detection basedon rolling circle amplification (RCA). The capture probe was first immobilized on theplane glass slide surface, which was chemically functionalized with a self-assemblingtriethoxysilylbutyraldehyde/N, N-dimethyl-N-octadecyl (3-aminopropyl)trimethoxysilyl chloride (TEA/DMOAP) film and then was used to capture the RCAproducts. In the presence of mutant target, the RCA product is a long single-strandedDNA molecule containing tandem repeats complementary to the original circlesequence. The bound DNA duplexes and long RCA products on the LC sensor substratesurface can greatly change the surface topology and further induce ahomeotropic-to-tiled transition of the LC molecules surrounding them and produce ashift change in the optical response of LC. This method is simple and label-freeincorporation the high selectivity of RCA technology with the convenience and lowcost of LC assay.In chapter4: To enhance the capability of metal ions disturbing the orientation of LC, we designed a new label-free LC biosensor for the highly selective and sensitive detection of heavy metal ions. This strategy makes use of the target-induced DNA conformational change to enhance the disruption of target molecules for the orientation of LC leading to an amplified optical signal. The Hg2+ion, which possesses a unique property to bind specifically to two DNA thymine (T) bases, is used as a model heavy metal ion. In the presence of Hg2+, t he specific oligonucleotide probes form a conformational reorganization of the oligonucleotide probes from hairpin structure to duplex-like complexes. The duplex-like complexes are then bound on the triethoxysilylbutyraldehyde/N, N-dimethyl-N-octadecyl (3-aminopropyl) trimethoxysilyl chloride (TEA/DMOAP)-coated substrate modified with capture probes, which can greatly distort the orientational profile of LC, making the optical image of LC cell birefringent as a result. Theoptical signal of LC sensor has a visible change at the Hg2+concentration of low to0.1nM, showing good detection sensitivity. The cost-effective LC sensingmethod can translate the concentration signal of heavy metal ions in solution into the presence of DNA duplexes and is expected to be a sensitive detection platform for heavy metal ions and other small molecule monitors.In chapter5: A signal-on label-free liquid crystal biosensor was developed basedon adenosine triphosphate (ATP)-induced recombination of split aptamer chips. In thismethod, the single-strand ATP aptamer is split into two fragments. One of which isimmobilized on the TEA/DMOAP-coated substrate surface via covalent bind workingas the capture probe, the other part is used as the detection probe. In the presence ofATP, target-induced association of the two fragments thus increases the DNA densityat the substrate surface. This binding event can effectively disrupt the orientationalarrangement of LC, resulting in the corresponding changes of optical images. TheDNA complexes have been used as a signal amplifier to improve the sensitivity of thebiosensor, the detection limit of the proposed sensor is as low as10nM.In chapter6: A novel liquid crystal immunosensor based on the indirectcompetitive assay format was developed for the detection of β-indole acetic acid (IAA).IAA is a kind of small molecule antigen which has little effect on the orientation of theliquid crystal molecules, however its antibody with large molecular size has obviousinfluence on the alignment of liquid crystal molecules. IAA was tethered onto theAPTES/DMOAP self-assembled film surface on a glass slide via covalent bind. Thespecific binding of anti-IAA antibody and IAA induced the homeotropic-to-tiltedtransition of LC，resulting in the corresponding changes of optical images under thecrossed polarized light．The LC-based imaging method had a good signal-to-noise ratioand a clear distinction between positive and negative results． When the concentrationof analyte IAA exceeded a critical value(10-8M), the optical images showed a verystrong response． The proposed sensor exhibited high sensitivity and desirableselectivity，and was label free and easy in operation．...