Detection Of Biomolecules Using Aptamer-based Liquid Crystal Biosensor

It is well known that biosensors are composed of molecular recognition components and transducers.Traditional molecular recognition elements are mainly antibodies or enzymes,which are usually expensive and not easy to preserve for long periods of time.With the development of the SELEX（Systematic evolution of ligands by an exponential enrichment）technology,a growing number of aptamers were selected.And they have become very important components in the fabrication of the biosensors.Compared to the conventional antibodies,aptamers own obvious advantages,including simple synthesis,good stability,simplicity of modification and high flexibility for sensors.Therefore,the aptamers which are also called"artificial antibodies"have been widely used in the sensor fabrication.Liquid Crystal（LC）,a kind of special state of matter intermediate between solid and liquid,can serve as a novel signal amplification and transduction element because of its sensitive orientation response and unique birefringence.A novel biomolecular recognition platform—liquid crystal biosensor based on aptamers,can be fabricated by using aptamers with highly specificity as a molecular recognition probe and combining with sensitive optical response of LC molecules.The basic recognition principle is that the aptamers for the biomolecules are firstly modified onto the glass surface at certain concentration,allowing the homeotropic orientation of LCs on the substrate.The corresponding polarized images are uniform dark.In the presence of the target molecules on the glass substrate,the aptamers will specifically recognize them.In this case,the morphological and chemical composition of the glass substrate will change greatly,thus the orientational profile of LCs on the surface is changed,resulting in the corresponding polarized image of LCs birefringent.Then the target molecules can be recognized by the aptamer-based LC sensing platform.At present,there are few reports on the recognition of biomolecules based on the aptamer-based LC biosensor,and few people have studied it as a complete system.This research is to realize the optimization of this sensing platform,and to improve the sensitivity and specificity of recognition for the biological molecules on this sensing platform.This paper focuses on the following aspects:In chapter 2:The residues of antibiotics may bring potential harm to human beings,which has become a major problem at present.Kanamycin is a class of aminoglycoside antibiotics which is widely or even prevalently used.The author experimentally presents a sensitive and aptamer-based LC film for kanamycin detection.The relevant literatures have not been reported.N,N-dimethyl-N-（3-（trimethoxysilyl）propyl）-1-octadecanaminiuchloride （DMOAP）,Silane coupling agent（3-aminopropyl）triethoxysilane（APTES）and Glutaraldehyde（GA）were firstly chosen to be immobilized onto the substrate in the experiment.The aptamer decorated with amidogen was then modified on the substrate surface.In the presence of kanamycin,its interaction with the aptamers will result in the formation of G-quadruplexes.These will disrupt the orientated arrangement of the LCs on the surface to result in a color change of the polarized image.The method possesses high specificity and the detection limit is as low as 1 nM.As a result,this study is the first step of the optimization for this aptamer-based LC sensing platform.In chapter 3:The detection of pulmonary surfactant protein A（SP-A）was realized using the aptamer-based LC biosensor.On this sensing platform,the LCs are supported on the surface of glass slide modified with moderate SP-A aptamers,assuming a homeotropic orientation.Then the specific interaction between the SP-A aptamer and SP-A undergoes great changes of the topology on the surfaces.The changes of surfaces will disrupt the homeotropic orientation of LCs,bringing a color transition of polarized image.The detection limit is low to 5 nM.To make more in-depth analysis and research on the experimental results,we processed different polarized images（corresponding to different SP-A concentrations）and made the preliminary exploration of quantitative analysis by extracting the corresponding average gray values（that is,the color image of the average brightness values）of the polarized images.The result indicated that the average brightness value of the polarized image increased with the increase of SP-A concentration.This study presented a new idea for analysis of the detection results from LC biosensors.In chapter 4:To improve the recognition performance of the sensing platform,gold nanoparticles were blended with desired concentrations of the target molecules.Ractopamine（RAC）detection was performed on the sensing platform based on gold nanoparticle（AuNPs）signal enhancement.The RAC aptamers were immobilized on the self-assembled film of the glass slide to be the specific recognition probes for RAC,also allowing the homeotropic orientation of LCs.In the presence of RAC,AuNPs-RAC-Aptamer conjugates will form on the sensing interface of the biosensor,which can remarkably change the orientational profile of LCs,resulting in the corresponding polarized image of LCs birefringent.The limit of RAC detection is as low as 1 pmol?L^-1(equivalent to 0.3 pg?mL^-1).It is important to note that the nanoparticles need not be labeled onto the aptamers like the usual method.This study provides a simple,sensitive and novel method for ractopamine detection.In chapter 5:To decrease the difficulty of the preparation for the sensing substrate,we further investigated the use of single strand DNA（ss DNA）as a homeotropic orientation element of liquid crystals for detection of DNA hybridization.In this section,ssDNA of the appropriate length and concentration along with the triethoxysilylbutyraldehyde（TEA）was coated on the glass slide,assuming the suitable molecular film of proper surface coverage formed on the surface of the biosensor,and then they were used to realize the DNA hybridization.When a certain coverage of ssDNA was modified on the substrate of the biosensor,the LCs assumed homeotropic orientation.Otherwise,in the presence of complementary strand,the ssDNA hybridized with it and then the double-stranded DNA formed on the substrate.It disrupted the homeotropic orientation of LCs,resulting in the changes of the polarized image from black to color.This method is label-free,owing a high sensitivity and specificity,which will open up a new way for the recognition technique of liquid crystal biosensor based on aptamers.